Interleukin-2/interleukin-2 receptor alpha fusion proteins and methods of use

ABSTRACT

Disclosed herein are fusion proteins comprising: (a) a first polypeptide comprising Interleukin-2 (IL2); and (b) a second polypeptide, fused in frame to the first polypeptide, wherein the second polypeptide comprises an extracellular domain of Interleukin-2 Receptor alpha (IL2Rα), wherein IL2 or IL2Rα comprises at least one fewer glycosylation site compared to native IL2 or native IL2Rα. Methods of production and methods of therapeutic use of the fusion proteins are also disclosed.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 16/366,838, filed Mar. 27, 2019, which claims the benefit of U.S.Provisional Application No. 62/649,379, filed Mar. 28, 2018, which isincorporated by reference herein in its entirety.

2. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing in ASCIItext file (Name: 3338_1000002_Seqlisting_ST25.txt; Size: 354,585 bytes;and Date of Creation: Jun. 7, 2019) filed with the application is hereinincorporated by reference in its entirety.

3. FIELD

The presently disclosed subject matter generally relates to methods andcompositions for modulating the immune response employing anInterleukin-2/Interleukin-2 Receptor-alpha fusion protein.

4. BACKGROUND OF THE DISCLOSURE

Interleukin-2 (IL2 or IL-2) is a biologic cytokine that regulates keyaspects of the immune system. IL2 has been used in attempts to boostimmune responses in patients with cancer, inflammatory disease, or anautoimmune disease. IL2 is a potent T cells growth factor that promotesimmune responses, including clonal expansion of antigen-activated Tcells, drives development of CD4+ T-helper (Th)1 and Th2 cells,terminally differentiates CD8+ cytotoxic T lymphocytes (CTLs), andopposes development of CD4+ Th17 and T-follicular helper (Tfh) cells.IL2 also shapes T cell memory recall responses.

Clinical trials have harnessed the T cell activating properties of IL2in patients with cancer and HIV/AIDS by infusion of high doses of IL2(typically >500,000 units/kg, repeatedly) to boost T and NK cells.Indeed, IL2 was approved by the FDA for use in patients with melanomaand renal cell carcinoma as some (approximately 5%) exhibited completeremissions. Nevertheless, response rates were low in these and othercancers while this therapy was accompanied by severe toxicity. Just thesame, IL2 was deemed to be not effective in promoting immunity inHIV/AIDS patients. The poor efficacy of high dose IL2 in these settingsis due in part to the accompanying expansion of Tregs.

More recently, lower doses of IL2 have been used to selectively boosttolerance to suppress unwanted immune responses associated withautoimmune-like attack of self tissues. These low doses of IL2 have notshown any signs of enhancing or re-activation of autoreactive T cells.Preclinical studies showed that low IL2R signaling selectively promotedkey activities of Tregs but not T effector (Teff) cells and thattreatment of mice with low levels of IL2 prevented autoimmunity.Currently, a number of patients with hyperactive immune responses havebeen treated with low-dose IL2 (0.5-2 million units, periodically). Theexperience thus far has been that this therapy is safe, with noindication of reactivation of auto-aggressive T cells, while Tregsincrease in nearly all patients, which is often accompanied by clinicalimprovement. Nevertheless, IL2 has important drawbacks as a therapeutic,including a very short-half life in vivo, which limits its efficacy, andtoxicity at high doses. For these reasons new IL2 biologics are neededhaving improved pharmacokinetics and durability of responses for use.

5. SUMMARY OF THE DISCLOSURE

The present disclosure includes a fusion protein comprising (a) a firstpolypeptide comprising an Interleukin-2 (IL2) polypeptide; and (b) asecond polypeptide comprising an extracellular domain of anInterleukin-2 Receptor alpha (IL2Rα) polypeptide; wherein (i) theextracellular domain of the IL2Rα polypeptide has at least one fewerglycosylation compared to the extracellular domain of native IL2Rα (SEQID NO:7); and/or (ii) the IL2 polypeptide has at least one fewerglycosylation compared to native IL2 (SEQ ID NO:2); and wherein thefusion protein has IL2 activity. In some embodiments, the extracellulardomain of the IL2Rα polypeptide has at least one fewer glycosylation, atleast two fewer glycosylations, at least three fewer glycosylations, atleast four fewer glycosylations, at least five fewer glycosylations, atleast six fewer glycosylations, at least seven fewer glycosylations, atleast eight fewer glycosylations, or at least nine fewer glycosylationscompared to the extracellular domain of native IL2Rα (SEQ ID NO:7). Insome embodiments, the IL2 polypeptide has at least one fewerglycosylation compared to native IL2 (SEQ ID NO:2).

In some embodiments, the fusion protein comprises a first polypeptide,wherein the first polypeptide comprises an amino acid sequence at leastabout 60%, at least about 70%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99% or about 100%identical to SEQ ID NO:2. In some embodiments, the fusion proteincomprises a second polypeptide, wherein the second polypeptide comprisesan amino acid sequence at least about 60%, at least about 70%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or about 100% identical to SEQ ID NO:12.

In some embodiments, the extracellular domain of the IL2Rα polypeptidehaving at least one fewer glycosylation comprises a mutation thatremoves a glycosylation. In some embodiments, the mutation removes anO-glycosylation and/or an N-glycosylation. In some embodiments, themutation removes an O-glycosylation. In some embodiments, the mutationremoves an N-glycosylation.

In some embodiments, the mutation in the extracellular domain of theIL2Rα polypeptide is a deletion of amino acids 167 to 219, amino acids168 to 219, amino acids 169 to 219, amino acids 170 to 219, amino acids171 to 219, amino acids 172 to 219, amino acids 173 to 219, amino acids174 to 219, amino acids 175 to 219, amino acids 176 to 219, amino acids177 to 219, amino acids 178 to 219, amino acids 179 to 219, amino acids180 to 219, amino acids 181 to 219, amino acids 182 to 219, amino acids183 to 219, amino acids 184 to 219, amino acids 185 to 219, amino acids186 to 219, amino acids 187 to 219, amino acids 188 to 219, amino acids189 to 219, amino acids 190 to 219, amino acids 191 to 219, or aminoacids 192 to 219, corresponding to SEQ ID NO:7. In some embodiments, themutation is a deletion of amino acids from 167, 169, 171 through 192 to219, corresponding to SEQ ID NO:7. In some embodiments, the mutationdoes not include a deletion of 170 to 219, corresponding to SEQ ID NO:7.In some embodiments, the second polypeptide is SEQ ID NO:11. In someembodiments, the second polypeptide is SEQ ID NO:12.

In some embodiments, the mutation is one or more substitutions of anamino acid that is glycosylated with an amino acid that is notglycosylated. In some embodiments, the mutation is one or moresubstitutions of an amino acid that allows glycosylation at a nearbyamino acid with an amino acid that does not allow glycosylation at thenearby amino acid. In some embodiments, the one or more substitutionsare at amino acid N49, amino acid N68, amino acid T74, amino acid T85,amino acid T197, amino acid T203, amino acid T208, and amino acid T216,or any combination thereof, wherein the amino acid locations correspondto SEQ ID NO:7. In some embodiments, the one or more substitutions arefrom asparagine to an amino acid selected from the group consisting ofalanine, threonine, serine, arginine, aspartic acid, glutamine, glutamicacid, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, tryptophan, tyrosine, and valine. In someembodiments, the one or more substitutions are from threonine to anamino acid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine, and valine.

In some embodiments, the one of the substitutions is amino acid N49. Insome embodiments, N49 is mutated to an amino acid selected from thegroup consisting of alanine, threonine, serine, arginine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, andvaline.

In some embodiments, the one the substitutions is amino acid N68. Insome embodiments, N68 is mutated to an amino acid selected from thegroup consisting of alanine, threonine, serine, arginine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, andvaline.

In some embodiments, the one of the substitutions is amino acid T74. Insome embodiments, T74 is mutated to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan,tyrosine, and valine.

In some embodiments, the one of the substitutions is amino acid T85. Insome embodiments, T85 is mutated to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan,tyrosine, and valine.

In some embodiments, the one of the substitutions is amino acid T197. Insome embodiments, T197 is mutated to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan,tyrosine, and valine.

In some embodiments, the one of the substitutions is amino acid T203. Insome embodiments, T203 is mutated to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan,tyrosine, and valine.

In some embodiments, the one of the substitutions is amino acid T208. Insome embodiments, T208 is mutated to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan,tyrosine, and valine.

In some embodiments, the one of the substitutions is amino acid T216. Insome embodiments, T216 is mutated to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan,tyrosine, and valine.

In some embodiments, the one or more substitutions are at amino acidS50, amino acid S51, amino acid T69, amino acid T70, amino acid C192, orany combination thereof, wherein the amino acid locations correspond toSEQ ID NO:7.

In some embodiments, the one of the substitutions is at amino acid S50.In some embodiments, S50 is mutated to proline.

In some embodiments, the one of the substitutions is amino acid S51. Insome embodiments, S51 is mutated to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan,tyrosine, and valine.

In some embodiments, the one of the substitution is amino acid T69. Insome embodiments, T69 is mutated to proline.

In some embodiments, the one of the substitutions is amino acid T70. Insome embodiments, T70 is mutated to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, threonine,tryptophan, tyrosine, and valine.

In some embodiments, the one of the substitutions is amino acid C192. Insome embodiments, C192 is mutated to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

In some embodiments, the IL2 polypeptide having at least one fewerglycosylation comprises a mutation that removes a glycosylation. In someembodiments, the mutation is one or more substitutions of an amino acidthat is glycosylated with an amino acid that is not glycosylated. Insome embodiments, the mutation is one or more substitutions of an aminoacid that allows glycosylation at a nearby amino acid with an amino acidthat does not allow glycosylation at the nearby amino acid. In someembodiments, the one or more substitutions are from an alanine to anamino acid selected from the group consisting of arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine. In some embodiments, theone or more substitutions are from a threonine to an amino acid selectedfrom the group consisting of alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline,tryptophan, tyrosine, and valine. In some embodiments, the one or moresubstitutions are from a cysteine to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. In some embodiments, the one or moresubstitutions are from a cysteine to a serine. In some embodiments, theone or more substitutions are from a cysteine to an alanine. In someembodiments, the one or more substitutions are from a cysteine to avaline.

In some embodiments, the one or more substitutions are at amino acid T3compared to corresponding to SEQ ID NO:2. In some embodiments, one ofthe substitutions is at amino acid C125. In some embodiments, thesubstitution at amino acid C125 is selected from the group consisting ofC125S, C125A, and C125V.

In some embodiments, the mutation is a deletion. In some embodiments,the deletion is at amino acid A1.

In some embodiments, the fusion protein is deglycosylated enzymaticallyor chemically. In some embodiments, the fusion protein is deglycosylatedby alkali, hydrazinolysis, PNGase F, Endo H, O-glycosidase, or anycombination thereof.

In some embodiments, the fusion protein further comprises a linker fusedin frame between the first polypeptide and the second polypeptide. Insome embodiments, the linker is a glycine/serine linker. In someembodiments, the glycine/serine linker comprises an amino acid sequenceof (GS)_(n), (GGS)_(n), (GGGS)_(n), (GGGGS)_(n), or (GGGGS)_(n), whereinn is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, the glycine/serine linker comprises the amino acid sequenceof (GGGS)₃.

In some embodiments, the fusion protein further comprises a heterologousmoiety fused to the first polypeptide and/or the second polypeptide. Insome embodiments, the heterologous moiety is a half-life extendingmoiety. In some embodiments, the heterologous moiety comprises anon-polypeptide moiety. In some embodiments, the heterologous moietycomprises a polypeptide. In some embodiments, the heterologous moietycomprises albumin, an immunoglobulin constant region or a portionthereof, an immunoglobulin-binding polypeptide, an immunoglobulin G(IgG), albumin-binding polypeptide (ABP), a PASylation moiety, aHESylation moiety, XTEN, a PEGylation moiety, an Fc region, and anycombination thereof.

In some embodiments, the fusion protein is more stable than thepolypeptide consisting of SEQ ID NO:2 or SEQ ID NO:13. In someembodiments, the fusion protein has one or more properties selected fromthe group consisting of (i) increased thermodynamic stability comparedto a reference protein; (ii) increased TM compared to a referenceprotein; (iii) increased resistant to degradation compared to areference protein; (iv) increased resistance to modifications comparedto a reference protein; (v) increased stability in vivo compared to areference protein; and (vi) any combination thereof, wherein thereference protein comprises (i) a first polypeptide comprising anInterleukin-2 (IL2) polypeptide; and (b) a second polypeptide comprisingan extracellular domain of an Interleukin-2 Receptor alpha (IL2Rα)polypeptide; and has at least one fewer glycosylation compared to thefusion protein.

In some embodiments, the fusion protein is a monomer. In someembodiments, the fusion protein is a dimer. In some embodiments, thedimer comprises two monomers, and the monomers are associated with eachother via covalent bonds. In some embodiments, the dimer comprises twomonomers, and the monomers are associated via non-covalent bonds.

In some embodiments, the fusion protein has one or more pharmacokineticproperties selected from the group consisting of an increased half-life,increased C_(max), increased AUC, increased C_(min), decreasedclearance, improved bioavailability, and any combination thereof,compared to the pharmacokinetic property of the polypeptide consistingof SEQ ID NO:2 or SEQ ID NO:13. In some embodiments, the fusion proteinhas an extended half-life. In some embodiments, the extended half-lifeis at least about 1.5 fold, at least about 2 fold, at least about 3fold, at least about 4 fold, at least about 5 fold, at least about 6fold, at least about 7 fold, at least about 8 fold, at least about 9fold, at least about 10 fold, at least about 11 fold, at least about 12fold, at least about 13 fold, at least about 14 fold, at least about 15fold, at least about 16 fold, at least about 17 fold, at least about 18fold, at least about 19 fold, at least about 20 fold, at least about 21fold, or at least about 22 fold compared to the half-life of apolypeptide consisting of SEQ ID NO:2 or SEQ ID NO:13.

In some embodiments, disclosed herein is one or more fusion proteins. Insome embodiments, provided herein is a composition comprising one ormore fusion proteins disclosed herein.

In some embodiments, disclosed herein is a nucleic acid that encodes anyone of the fusion proteins disclosed herein. In some embodiments,disclosed herein is a vector comprising a nucleic acid that encodes anyone of the fusion proteins disclosed herein. In some embodiments,disclosed herein is a host cell comprising the nucleic acid that encodesany one of the fusion proteins disclosed herein. In some embodiments,the host cell is a eukaryotic cell. In some embodiments, the host cellis selected from the group consisting of a mammalian cell, an insectcell, a yeast cell, a transgenic mammalian cell, and a plant cell. Insome embodiments, the host cell is a mammalian cell. In someembodiments, the host cell is a prokaryotic cell. In some embodiments,the prokaryotic cell is a bacterial cell.

In some embodiments, provided herein is a pharmaceutical compositioncomprising (a) a fusion protein disclosed herein, a compositiondisclosed herein, a nucleic acid disclosed herein, a vector disclosedherein, or a host cell disclosed herein; and (b) a pharmaceuticallyacceptable excipient.

In some embodiments, provided herein is a kit comprising the fusionprotein disclosed herein, the composition disclosed herein, the nucleicacid disclosed herein, the vector disclosed herein, or the host celldisclosed herein and instructions for administering the fusion proteinto a subject in need thereof.

In some embodiments, provided herein is a method of producing the fusionprotein disclosed herein, comprising: culturing the host cell disclosedherein under suitable conditions and recovering the fusion protein. Insome embodiments, the host cell is a eukaryotic cell or a prokaryoticcell. In some embodiments, the host cell is a mammalian cell, an insectcell, a fungal cell, a plant cell, a transgenic mammalian cell, or abacterial cell. In some embodiments, the host cell is selected from thegroup consisting of a CHO cell, a HEK 293 cell, a NS0 cell, a Per C6cell, a BHK cell, and a COS cell. In some embodiments, the bacterialcell is Escherichia coli.

In some embodiments, provided herein is a method of treating a diseaseor disorder a subject in need thereof, comprising administering to thesubject an effective amount of a fusion protein disclosed herein, acomposition disclosed herein, a nucleic acid disclosed herein, a vectordisclosed herein, a host cell disclosed herein, or a pharmaceuticalcomposition disclosed herein. In some embodiments, the disease ordisorder is cancer. In some embodiments, the cancer is a bladder cancer,breast cancer, uterine cancer, endometrial carcinoma, ovarian cancer,colorectal cancer, colon cancer, head and neck cancer, lung cancer,stomach cancer, germ cell cancer, bone cancer, squamous cell cancer,skin cancer, neoplasm of the central nervous system, lymphoma, leukemia,sarcoma, virus-related cancer, small-cell lung cancer, non-small celllung cancer, gastrointestinal cancer, Hodgkin's or non-Hodgkin'slymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer,ovarian cancer, liver cancer, myeloma, salivary gland carcinoma, kidneycancer, basal cell carcinoma, melanoma, prostate cancer, vulval cancer,thyroid cancer, testicular cancer, esophageal cancer, or head or neckcancer, and any combinations thereof.

In some embodiments, the disease or disorder is an inflammatory diseaseor an autoimmune disease. In some embodiments, the inflammatory diseaseor an autoimmune disease is selected from the group consisting of type 1diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease,systemic lupus erythematous, lupus nephritis, cutaneous lupus, juvenileidiopathic arthritis, Crohn's disease, ulcerative colitis or systemicsclerosis, graft versus host disease, psoriasis, alopecia areata,HCV-induced vasculitis, Sjogren's syndrome, Pemphigus, AnkylosingSpondylitis, Behcet's Disease, Wegener's Granulomatosis, Takayasu'sDisease, Autoimmune Hepatitis, Sclerosing Cholangitis, Gougerot-sjögren,and Macrophage Activation Syndrome.

In some embodiments, the disease or disorder is an infectious disease.In some embodiments, the infectious disease is caused by a pathogenicvirus. In some embodiments, the pathogenic virus is selected from thegroup consisting of human immunodeficiency virus (HIV), hepatitis A,hepatitis B, hepatitis C, herpes virus, adenovirus, influenza virus,flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, human T-lymphotropic (HTL)virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabiesvirus, John Cunningham (JC) virus and arboviral encephalitis virus. Insome embodiments, the infectious disease is caused by pathogenicbacteria. In some embodiments, the pathogenic bacteria is selected fromthe group consisting of Chlamydia, rickettsial bacteria, mycobacteria,staphylococci, streptococci, pneumonococci, meningococci and gonococci,Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria,Salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague,leptospirosis, and Lymes disease bacteria. In some embodiments, theinfectious disease is caused by pathogenic fungi. In some embodiments,the pathogenic bacteria is selected from the group consisting of Candida(albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans,Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia,rhizopus), Sporothrix schenkii, Blastomyces dermatitidis,Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasmacapsulatum. In some embodiments, the infectious disease is caused bypathogenic parasite. In some embodiments, the pathogenic parasite isselected from the group consisting of Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, and Nippostrongylus brasiliensis.

In some embodiments, the methods disclosed herein further compriseadministering to the subject a second agent. In some embodiments, thesecond agent is a PD-1 antagonist, a CTLA-4 antagonist, a TIM3antagonist, a GITR antagonist, a KIR antagonist, a LAG3 antagonist, orany combination thereof. In some embodiments, the second agent is ananti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, ananti-TIM3 antibody, an anti-KIR antibody, an anti-GITR antibody, ananti-LAG3 antibody, or any combination thereof. In some embodiments, thesecond agent is a cytokine inhibitor. In some embodiments, the cytokineinhibitor targets one or more of IL-6, IL-10, TGF-β, VEGF, IFN-γ, or anycombination thereof.

In some embodiments, the fusion protein is administered via a topical,epidermal mucosal, intranasal, oral, vaginal, rectal, sublingual,topical, intravenous, intraperitoneal, intramuscular, intraarterial,intrathecal, intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural orintrasternal route.

6. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that molecular mass of SEQ ID NO:16 is 93 kDa as measuredby SEC/MALS static light scattering. Theoretical mass of the polypeptidechain alone is 37,812 Da reduced. The observed solution massdemonstrates it exists as a homogeneous homo-dimer with about 19% of themass due to glycosylation.

FIG. 2 shows prototypical binding of the IL2-CD25 fusion protein (SEQ IDNO: 16) to human sCD25 using surface plasmon resonance measurements.

FIG. 3 shows serum concentration-time profiles of indicated fusionproteins in Balb/C mice following a 0.5 mg/kg single intravenous (IV) orsubcutaneous (SC) dose. Each time-point represents a mean of samplesfrom 3 mice. Error bars represent standard deviation.

FIG. 4 shows serum concentration-time profiles of indicated fusionproteins in Balb/C mice following a 0.5 mg/kg single intravenous (IV) orsubcutaneous (SC) dose. Each time-point represents a mean of samplesfrom 3 mice. Error bars represent standard deviation.

FIG. 5 shows serum concentration-time profiles of hIL2-CD25 (22-212) incynomolgus monkey following a 0.075 mg/kg single subcutaneous (SC) dose.Each time-point represents a mean of samples from 3 monkeys. Error barsrepresent standard deviation.

FIG. 6 shows the activity of IL2-CD25 fusion proteins to induce STAT5phosphorylation in human PBMCs from a representative donor. Cells weregated on Treg (CD4⁺, foxp3⁺, CD25⁺) or Tconv (CD4⁺, foxp3⁻), CD8⁺, andNK cells (CD3⁻, CD56⁺) and the percent of cells which stained positivefor pSTAT5 after incubation was quantitated. The EC₅₀ for pSTAT5induction in Treg determined from these experiments was 10 ng/ml, 4.4ng/ml, and 4.6 ng/ml for hIL2-CD25(22-240), hIL2-CD25(22-212), andhIL2-CD25(22-187) respectively.

FIG. 7 shows the ability of truncated fusions proteins to stimulate IL2Rin whole blood resulting in induction of phosphorylated STAT5 in variouscell types. IL2R signaling was detected by measuring by flow cytometryafter intracellular staining for pSTAT5 and determining the percent ofcells staining positive for pSTAT5 in the whole blood mixture. Thepotency of hIL2-CD25(22-240) was compared to hIL2-CD25(22-212) in arepresentative donor. Protein was titrated in human whole blood and theintensity of intracellular pSTAT5 staining measured by flow cytometry.EC50 for pSTAT5 induction in Treg was 22 ng/ml for hIL2-CD25(22-240) and36 ng/ml for hIL2-CD25(22-212).

FIG. 8A-FIG. 8C show the similar ability of hIL2-CD25(22-240),hIL2-CD25(22-212) and hIL2-CD25(22-184) to increase T cells in mice witha humanized immune system. Administration (s.c.) of fusion protein toNSG-huCD34 engrafted mice was performed every third day for three doses.Analysis of the Treg (FIG. 8A), CD8 (FIG. 8B), and NK cells (FIG. 8C)from the spleen of dosed mice was determined by flow cytometry.

FIG. 9A and FIG. 9B show phosphorylation of STAT5 after 15 min in amixed cell population including in PBMCs (FIG. 9A) or whole bloodconcentration (FIG. 9B) with hIL2-CD25(22-212) (SEQ ID NO:16).

FIG. 10 shows LC-MS/MS based analysis of (G3S)3 linker stability inIL2(21-153)-(G3S)3-CD25(22-212) in human or mouse serum in vitro. Peakarea ratio of IL2 and CD25 using LC-MS/MS is reported after captureusing anti-IL2 antibody.

FIG. 11 shows LC-MS/MS based analysis of (G3S)3 linker stability inIL2(21-153)-(G3S)3-CD25(22-212) in serum of monkeys after a singlesubcutaneous dose of 0.075 mg/kg. Peak area ratio of IL2 and CD25 usingLC-MS/MS is reported after capture using anti-IL2 antibody.

7. DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure is described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the disclosure are shown. Indeed, these disclosures canbe embodied in many different forms and should not be construed aslimited to the embodiments set forth herein; rather, these embodimentsare provided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the disclosures set forthherein will come to mind to one skilled in the art to which thesedisclosures pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the disclosures are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

7.1 Overview

Various methods and compositions are provided which can be employed tomodulate the immune system. Compositions include a fusion proteincomprising: (a) a first polypeptide comprising Interleukin-2 (IL2)polypeptide; and (b) a second polypeptide comprising an extracellulardomain of Interleukin-2 Receptor alpha (IL2Rα) polypeptide; wherein (i)the extracellular domain of the IL2Rα polypeptide has at least one fewerglycosylation compared to the extracellular domain of native IL2Rα (SEQID NO:7); and/or (ii) the IL2 polypeptide has at least one fewerglycosylation compared to native IL2 (SEQ ID NO:2); and wherein thefusion protein has IL2 activity.

The present disclosure also describes a nucleotide encoding the fusionprotein disclosed herein, a vector comprising the nucleotide, a hostcell comprising the nucleotide, and a composition comprising the fusionprotein, the nucleotide, the vector, or the host cell. The disclosure isalso directed to methods of making the fusion protein, the nucleotide,the vector, the host cell, or the composition or methods of using thefusion protein, the nucleotide, the vector, the host cell, or thecomposition.

7.2 Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleotide sequence,” is understood torepresent one or more nucleotide sequences. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Similarly, the word “or” is intended to include “and” unless the contextclearly indicates otherwise. It is further to be understood that allbase sizes or amino acid sizes, and all molecular weight or molecularmass values, given for nucleic acids or polypeptides are approximate,and are provided for description.

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects ofthe disclosure, which can be had by reference to the specification as awhole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

The term “about” is used herein to mean approximately, roughly, around,or in the regions of. When the term “about” is used in conjunction witha numerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. Thus, “about 10-20”means “about 10 to about 20.” In general, the term “about” can modify anumerical value above and below the stated value by a variance of, e.g.,10 percent, up or down (higher or lower).

As used herein, “Interleukin-2”, “IL2”, or “IL-2” refers to any nativeor recombinant IL2 from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), anddomesticated or agricultural mammals unless otherwise indicated. Theterm encompasses unprocessed IL2, as well as, any form of IL2 thatresults from processing in the cell (i.e., the mature form of IL2). Theterm also encompasses naturally occurring variants and fragments of IL2,e.g. splice variants or allelic variants, and non-naturally occurringvariants that have IL2 activity of the naturally occurring IL2.

Additional nucleic acid and amino acid sequences for IL2 are known. See,for example, GenBank Accession Nos: Q7JFM2 (Aotus lemurinus(Gray-bellied night monkey)); Q7JFM5 (Aotus nancymaae (Ma's nightmonkey)); P05016 (Bas taurus (Bovine)); Q29416 (Canisfamiliaris (Dog)(Canis lupus familiaris)); P36835 (Capra hircus (Goat)); and, P37997(Equus caballus (Horse)).

Biologically active fragments and variants of IL2 retain IL2 activity.The phrase “biological activity of IL2” or “IL2 activity” refers to oneor more of the biological activities of IL2, including but not limitedto, the ability to stimulate IL2 receptor bearing lymphocytes. Suchactivity can be measured both in vitro and in vivo. IL2 is a globalregulator of immune activity and the effects seen here are the sum ofsuch activities. For example, it regulates survival activity (Bcl-2),induces T effector activity (IFN-gamma, Granzyme B, and Perforin),and/or promotes T regulatory activity (FoxP3).

Biologically active variants of IL2 are known. See, for example, USApplication Publications 20060269515 and 20060160187 and WO 99/60128.

The term “CD25,” “IL2 receptor α,” “IL2Rα,” or “IL2Ra” as used herein,refers to any native or recombinant IL2Rα from any vertebrate source,including mammals such as primates (e.g. humans) and rodents (e.g., miceand rats) and domesticated or agricultural mammals unless otherwiseindicated. The term also encompasses naturally occurring variants ofIL2Rα, e.g., splice variants or allelic variants, or non-naturallyoccurring variants that have IL2Rα activity. Human IL2 exerts itsbiological effects via signaling through its receptor system, IL2R. IL2and its receptor (IL2R) are required for T-cell proliferation and otherfundamental functions which are crucial for the immune response. IL2Rconsists of 3 non-covalently linked type I transmembrane proteins, whichare the alpha (p55), beta (p75), and gamma (p65) chains. The human IL2Ralpha chain contains an extracellular domain of 219 amino acids, atransmembrane domain of 19 amino acids, and an intracellular domain of13 amino acids. The secreted extracellular domain of IL2R alpha (IL2R-α)can be employed in the fusion proteins describe herein.

Nucleic acid and amino acid sequences for IL2Rα are known. See, forexample, GenBank Accession Nos: NP_001030597.1 (Pan troglodytes);NP_001028089.1 (Macaca mulatta); NM_001003211.1 (Canis lupus);NP_776783.1 (Bos taurus); NP_032393.3 (Mus musculus); and, NP_037295.1(Rattus norvegicus).

Biologically active fragments and variants of the extracellular domainof IL2Rα are also provided. Such IL2Rα extracellular domain activevariants or fragments will retain the IL2Rα extracellular domainactivity. The phrase “biological activity of the IL2Rα extracellulardomain” refers to one or more of the biological activities ofextracellular domain of IL2Rα, including but not limited to, the abilityto bind to IL2 and/or enhance intracellular signaling in IL2 receptorresponsive cells. Non-limiting examples of biologically active fragmentsand variants of the IL2Rα are disclosed, for example, in Robb et al.,Proc. Natl. Acad. Sci. USA, 85:5654-5658, 1988. In some embodiments, thebiologically active fragments and variants of the IL2Rα disclosed hereincomprise at least one fewer glycosylation compared to the extracellulardomain of native IL2Rα.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

A “polypeptide” refers to a chain comprising at least two consecutivelylinked amino acid residues, with no upper limit on the length of thechain. One or more amino acid residues in the protein can contain amodification such as, but not limited to, glycosylation, phosphorylationor disulfide bond formation. A “protein” or “fusion protein” cancomprise one or more polypeptides.

Also included in the present disclosure are fragments or variants ofpolypeptides, and any combination thereof. The term “fragment” or“variant” when referring to polypeptide binding domains or bindingmolecules of the present disclosure include any polypeptides whichretain at least some of the properties (e.g., IL2 binding activity forIL2Rα) of the reference polypeptide. Fragments of polypeptides includeproteolytic fragments, as well as deletion fragments, but do not includethe naturally occurring full-length polypeptide (or mature polypeptide).Variants of polypeptide binding domains or binding molecules of thepresent disclosure include fragments as described above, and alsopolypeptides with altered amino acid sequences due to amino acidsubstitutions, deletions, or insertions. Variants can be naturally ornon-naturally occurring. Non-naturally occurring variants can beproduced using art-known mutagenesis techniques. Variant polypeptidescan comprise conservative or non-conservative amino acid substitutions,deletions or additions.

As stated above, polypeptide variants include, e.g., modifiedpolypeptides. Modifications include, e.g., acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation (Mei et al., Blood 116:270-79 (2010)), proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

As used herein, an “corresponding to,” “amino acid corresponding to,”“site corresponding to,” or “equivalent amino acid” in a protein ornucleotide sequence is identified by alignment to maximize the identityor similarity between a first protein sequence, e.g., an IL2 sequence,and a second protein sequence, e.g., a second IL2 sequence. The numberused to identify an equivalent amino acid in a second protein sequenceis based on the number used to identify the corresponding amino acid inthe first protein sequence. In some embodiments, the term “correspondingto” refers to the relationship of a mutation at one or more amino acidsin a polypeptide or one or more nucleotides in a polynucleotide. By wayof a non-limiting example, a specific amino acid (e.g., S50) of apolynucleotide (e.g., SEQ ID NO:7) as disclosed herein refers to the50^(th) amino acid—a serine—in SEQ ID NO: 7.

As used herein the term “associated with” refers to a covalent ornon-covalent bond formed between a first amino acid chain and a secondamino acid chain. In one embodiment, the term “associated with” means acovalent, non-peptide bond or a non-covalent bond. This association canbe indicated by a colon, i.e., (:). In another embodiment, it means acovalent bond except a peptide bond. For example, the amino acidcysteine comprises a thiol group that can form a disulfide bond orbridge with a thiol group on a second cysteine residue. In mostnaturally occurring IgG molecules, the CH1 and CL regions are associatedby a disulfide bond and the two heavy chains are associated by twodisulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).Examples of covalent bonds include, but are not limited to, a peptidebond, a metal bond, a hydrogen bond, a disulfide bond, a sigma bond, api bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond,a dipolar bond, a Pi backbond, a double bond, a triple bond, a quadruplebond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation,aromaticity, hapticity, or antibonding. Non-limiting examples ofnon-covalent bond include an ionic bond (e.g., cation-pi bond or saltbond), a metal bond, an hydrogen bond (e.g., dihydrogen bond, dihydrogencomplex, low-barrier hydrogen bond, or symmetric hydrogen bond), van derWalls force, London dispersion force, a mechanical bond, a halogen bond,aurophilicity, intercalation, stacking, entropic force, or chemicalpolarity.

The term “comparable” as used herein means a compared rate or levelresulted from using, e.g., the fusion protein is equal to, substantiallyequal to, or similar to the reference rate or level. The term “similar”as used herein means a compared rate or level has a difference of nomore than 10% or no more than 15% from the reference rate or level. Theterm “substantially equal” means a compared rate or level has adifference of no more than 0.01%, 0.5% or 1% from the reference rate orlevel.

The term “expression” as used herein refers to a process by which apolynucleotide produces a gene product, for example, an RNA or apolypeptide.

A “fusion” or “fusion” protein comprises a first amino acid sequencelinked in frame to a second amino acid sequence with which it is notnaturally linked in nature. The amino acid sequences which normallyexist in separate proteins can be brought together in the fusionpolypeptide, or the amino acid sequences which normally exist in thesame protein can be placed in a new arrangement in the fusionpolypeptide, e.g., fusion of an IL2 protein with an IL2-Rα protein. Afusion protein is created, for example, by chemical synthesis, or bycreating and translating a polynucleotide in which the peptide regionsare encoded in the desired relationship. A fusion protein can furthercomprise a second amino acid sequence associated with the first aminoacid sequence by a covalent, non-peptide bond or a non-covalent bond.Upon transcription/translation, a single protein is made. In this way,multiple proteins, or fragments thereof can be incorporated into asingle polypeptide. “Operably linked” is intended to mean a functionallinkage between two or more elements. For example, an operable linkagebetween two polypeptides fuses both polypeptides together in frame toproduce a single polypeptide fusion protein. In a particular aspect, thefusion protein further comprises a third polypeptide which, as discussedin further detail below, can comprise a linker sequence.

An “Fc region” (fragment crystallizable region), “Fc domain,” or “Fc”refers to the C-terminal region of the heavy chain of an antibody thatmediates the binding of the immunoglobulin to host tissues or factors,including binding to Fc receptors located on various cells of the immunesystem (e.g., effector cells) or to the first component (C1q) of theclassical complement system. Thus, an Fc region comprises the constantregion of an antibody excluding the first constant region immunoglobulindomain (e.g., CH1 or CL). In IgG, IgA and IgD antibody isotypes, the Fcregion comprises two identical protein fragments, derived from thesecond (CH2) and third (CH3) constant domains of the antibody's twoheavy chains; IgM and IgE Fc regions comprise three heavy chain constantdomains (CH domains 2-4) in each polypeptide chain. The IgG isotype isdivided in subclasses in certain species: IgG1, IgG2, IgG3 and IgG4 inhumans, and IgG1, IgG2a, IgG2b and IgG3 in mice. For IgG, the Fc regioncomprises immunoglobulin domains CH2 and CH3 and the hinge between CH1and CH2 domains. Although the definition of the boundaries of the Fcregion of an immunoglobulin heavy chain might vary, as defined herein,the human IgG heavy chain Fc region is defined to stretch from an aminoacid residue D221 for IgG1, V222 for IgG2, L221 for IgG3 and P224 forIgG4 to the carboxy-terminus of the heavy chain, wherein the numberingis according to the EU index as in Kabat. The CH2 domain of a human IgGFc region extends from amino acid 237 to amino acid 340, and the CH3domain is positioned on C-terminal side of a CH2 domain in an Fc region,i.e., it extends from amino acid 341 to amino acid 447 or 446 (if theC-terminal lysine residue is absent) or 445 (if the C-terminal glycineand lysine residues are absent) of an IgG. As used herein, the Fc regioncan be a native sequence Fc, including any allotypic variant, or avariant Fc (e.g., a non-naturally-occurring Fc).

An “Fc receptor” or “FcR” is a receptor that binds to the Fc region ofan immunoglobulin. FcRs that bind to an IgG antibody comprise receptorsof the FcγR family, including allelic variants and alternatively splicedforms of these receptors. The FcγR family consists of three activating(FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA inhumans) and one inhibitory (FcγRIIB) receptor. Various properties ofhuman FcγRs are known in the art. The majority of innate effector celltypes co-express one or more activating FcγR and the inhibitory FcγRIIB,whereas natural killer (NK) cells selectively express one activating Fcreceptor (FcγRIII in mice and FcγRIIIA in humans) but not the inhibitoryFcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptorsand is considered equivalent to murine IgG2a with respect to the typesof activating Fc receptors that it binds to.

The terms “inserted,” “is inserted,” “inserted into,” or grammaticallyrelated terms, as used herein refers to the position of a heterologousmoiety (e.g., a half-life extending moiety) in a fusion polypeptiderelative to the analogous position in specified protein. As used hereinthe terms refer to the characteristics of the recombinant polypeptidedisclosed herein, and do not indicate, imply or infer any methods orprocess by which the fusion polypeptide was made.

“Heterologous” and “heterologous moiety” in reference to a polypeptideor polynucleotide is a polypeptide or polynucleotide that originatesfrom a different protein or polynucleotide. The additional components ofthe fusion protein can originate from the same organism as the otherpolypeptide components of the fusion protein, or the additionalcomponents can be from a different organism than the other polypeptidecomponents of the fusion protein. For instance, a heterologouspolypeptide can be synthetic, or derived from a different species,different cell type of an individual, or the same or different type ofcell of distinct individuals. In one aspect, a heterologous moiety is apolypeptide fused to another polypeptide to produce a fusion polypeptideor protein. In another aspect, a heterologous moiety is anon-polypeptide such as PEG conjugated to a polypeptide or protein.Non-limiting examples of heterologous moieties disclosed herein areglycine/serine linkers (e.g., GGGSGGGSGGGS (SEQ ID NO:71) (also noted as(Gly₃Ser)₃)).

A “native sequence Fc region” or “native sequence Fc” comprises an aminoacid sequence that is identical to the amino acid sequence of an Fcregion found in nature. Native sequence human Fc regions include anative sequence human IgG1 Fc region; native sequence human IgG2 Fcregion; native sequence human IgG3 Fc region; and native sequence humanIgG4 Fc region as well as naturally-occurring variants thereof. Nativesequence Fc include the various allotypes of Fcs (see, e.g., Jefferis etal. (2009) mAbs 1:1).

The term “EC₅₀” in the context of an in vitro or in vivo assay usingfusion protein refers to the concentration of a fusion protein thatinduces a response that is 50% of the maximal response, i.e., halfwaybetween the maximal response and the baseline.

“Conservative amino acid substitutions” refer to substitutions of anamino acid residue with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). In someembodiments, a predicted nonessential amino acid residue in IL2/IL2Rαfusion protein is replaced with another amino acid residue from the sameside chain family. Methods of identifying nucleotide and amino acidconservative substitutions which do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999);and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule can besingle-stranded or double-stranded, and can be cDNA.

As used herein, the term “regulatory region” refers to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding region, and whichinfluence the transcription, RNA processing, stability, or translationof the associated coding region. Regulatory regions may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures. If a coding region is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

A polynucleotide, which encodes a gene product, e.g., a polypeptide, caninclude a promoter and/or other transcription or translation controlelements operably associated with one or more coding regions. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can also be operably associated with a coding region to direct geneproduct expression.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions, which function in vertebrate cells, such as, but not limitedto, promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit ß-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

The term “percent sequence identity,” “percent identity,” “sequenceidentity,” or “identity” are used interchangeably and refers to thenumber of identical matched positions shared between two polynucleotideor polypeptide sequences over a comparison window, taking into accountadditions or deletions (i.e., gaps) that must be introduced for optimalalignment of the two sequences. A matched position is any position wherean identical nucleotide or amino acid is presented in both the targetand reference sequence. Gaps presented in the target sequence are notcounted since gaps are not nucleotides or amino acids. Likewise, gapspresented in the reference sequence are not counted since targetsequence nucleotides or amino acids are counted, not nucleotides oramino acids from the reference sequence.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

The percentage of sequence identity is calculated by determining thenumber of positions at which the identical amino-acid residue or nucleicacid base occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. The comparison ofsequences and determination of percent sequence identity between twosequences may be accomplished using readily available software both foronline use and for download. Suitable software programs are availablefrom various sources, and for alignment of both protein and nucleotidesequences. One suitable program to determine percent sequence identityis bl2seq, part of the BLAST suite of programs available from the U.S.government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between twosequences using either the BLASTN or BLASTP algorithm. BLASTN is used tocompare nucleic acid sequences, while BLASTP is used to compare aminoacid sequences. Other suitable programs are, e.g., Needle, Stretcher,Water, or Matcher, part of the EMBOSS suite of bioinformatics programsand also available from the European Bioinformatics Institute (EBI) atwww.ebi.ac.uk/Tools/psa.

Different regions within a single polynucleotide or polypeptide targetsequence that aligns with a polynucleotide or polypeptide referencesequence can each have their own percent sequence identity. It is notedthat the percent sequence identity value is rounded to the nearesttenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to80.2. It also is noted that the length value will always be an integer.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available atworldwideweb.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences described herein can further beused as a “query sequence” to perform a search against public databasesto, for example, identify related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acidmolecules described herein. BLAST protein searches can be performed withthe XBLAST program, score=50, word length=3 to obtain amino acidsequences homologous to the protein molecules described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See worldwideweb.ncbi.nlm.nih.gov.

The nucleic acids can be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids (e.g., the other parts of the chromosome) or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York (1987).

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”) In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, also included are other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The term “in vitro host cell” (or simply “host cell”), as used herein,is intended to refer to a cell that comprises a nucleic acid that is notnaturally present in the cell, and can be a cell into which arecombinant expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications can occur in succeeding generations due to eithermutation or environmental influences, such progeny cannot, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. Exemplary host cells include, butare not limited to, prokaryotic cells (e.g., E. coli), or alternatively,eukaryotic cells, for example, fungal cells (e.g., yeast cells such asSaccharomyces cerevisiae, Pichia pastoris, or Schizosaccharomycespombe), and various animal cells, such as insect cells (e.g., Sf-9) ormammalian cells (e.g., HEK293F, CHO, COS-7, NIH-3T3).

The phrase “immediately downstream of an amino acid” as used hereinrefers to position right next to the terminal carboxyl group of theamino acid. Similarly, the phrase “immediately upstream of an aminoacid” refers to the position right next to the terminal amine group ofthe amino acid. Therefore, the phrase “between two amino acids of aninsertion site” as used herein refers to a position in which aheterologous moiety (e.g., a half-life extending moiety) is insertedbetween two adjacent amino acids.

As used herein, “administering” refers to the physical introduction of acomposition comprising a therapeutic agent to a subject, using any ofthe various methods and delivery systems known to those skilled in theart. Different routes of administration for the IL2/IL2Rα fusion proteindescribed herein include intravenous, intraperitoneal, intramuscular,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intraperitoneal, intramuscular, intraarterial,intrathecal, intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

An “immune response” is as understood in the art, and generally refersto a biological response within a vertebrate against foreign agents orabnormal, e.g., cancerous cells, which response protects the organismagainst these agents and diseases caused by them. An immune response ismediated by the action of one or more cells of the immune system (forexample, a T lymphocyte, B lymphocyte, natural killer (NK) cell,macrophage, eosinophil, mast cell, dendritic cell or neutrophil) andsoluble macromolecules produced by any of these cells or the liver(including antibodies, cytokines, and complement) that results inselective targeting, binding to, damage to, destruction of, and/orelimination from the vertebrate's body of invading pathogens, cells ortissues infected with pathogens, cancerous or other abnormal cells, or,in cases of autoimmunity or pathological inflammation, normal humancells or tissues. An immune reaction includes, e.g., activation orinhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4⁺cell, a CD8⁺ T cell, or a Treg cell, or activation or inhibition of anyother cell of the immune system, e.g., NK cell.

An “immunomodulator” or “immunoregulator” refers to an agent, e.g., anagent targeting a component of a signaling pathway that can be involvedin modulating, regulating, or modifying an immune response.“Modulating,” “regulating,” or “modifying” an immune response refers toany alteration in a cell of the immune system or in the activity of suchcell (e.g., an effector T cell, such as a Th1 cell). More particularly,as used herein, the term “modulating” includes inducing, inhibiting,potentiating, elevating, increasing, or decreasing a given activity orresponse. Such modulation includes stimulation or suppression of theimmune system which can be manifested by an increase or decrease in thenumber of various cell types, an increase or decrease in the activity ofthese cells, or any other changes which can occur within the immunesystem. Both inhibitory and stimulatory immunomodulators have beenidentified, some of which can have enhanced function in a tumormicroenvironment. In some embodiments, the immunomodulator targets amolecule on the surface of a T cell. An “immunomodulatory target” or“immunoregulatory target” is a molecule, e.g., a cell surface molecule,that is targeted for binding by, and whose activity is altered by thebinding of, a substance, agent, moiety, compound or molecule.Immunomodulatory targets include, for example, receptors on the surfaceof a cell (“immunomodulatory receptors”) and receptor ligands(“immunomodulatory ligands”).

“Immunotherapy” refers to the treatment of a subject afflicted with, orat risk of contracting or suffering a recurrence of, a disease by amethod comprising inducing, enhancing, suppressing or otherwisemodifying the immune system or an immune response.

“Immuno stimulating therapy” or “immuno stimulatory therapy” refers to atherapy that results in increasing (inducing or enhancing) an immuneresponse in a subject for, e.g., treating cancer.

“Potentiating an endogenous immune response” means increasing theeffectiveness or potency of an existing immune response in a subject.This increase in effectiveness and potency can be achieved, for example,by overcoming mechanisms that suppress the endogenous host immuneresponse or by stimulating mechanisms that enhance the endogenous hostimmune response.

“T effector” (“T_(eff)”) cells refers to T cells (e.g., CD4⁺ and CD8⁺ Tcells) with cytolytic activities as well as T helper (Th) cells, e.g.,Th1 cells, which cells secrete cytokines and activate and direct otherimmune cells, but does not include regulatory T cells (Treg cells).Certain IL2/IL2Rα fusion proteins described herein activate T_(eff)cells, e.g., CD4⁺ and CD8⁺ T_(eff) cells and Th1 cells.

An increased ability to stimulate an immune response or the immunesystem, can result from an enhanced agonist activity of T cellco-stimulatory receptors and/or an enhanced antagonist activity ofinhibitory receptors. An increased ability to stimulate an immuneresponse or the immune system can be reflected by a fold increase of theEC₅₀ or maximal level of activity in an assay that measures an immuneresponse, e.g., an assay that measures changes in cytokine or chemokinerelease, cytolytic activity (determined directly on target cells orindirectly via detecting CD107a or granzymes) and proliferation. Theability to stimulate an immune response or the immune system activitycan be enhanced by at least 10%, 30%, 50%, 75%, 2 fold, 3 fold, 5 foldor more.

The terms “linked” and “fused” as used herein refers to a first aminoacid sequence or nucleotide sequence covalently or non-covalently joinedto a second amino acid sequence or nucleotide sequence, respectively.The first amino acid or nucleotide sequence can be directly joined orjuxtaposed to the second amino acid or nucleotide sequence oralternatively an intervening sequence can covalently join the firstsequence to the second sequence. The term “linked” means not only afusion of a first amino acid sequence to a second amino acid sequence atthe C-terminus or the N-terminus, but also includes insertion of thewhole first amino acid sequence (or the second amino acid sequence) intoany two amino acids in the second amino acid sequence (or the firstamino acid sequence, respectively). In one embodiment, the first aminoacid sequence is linked to a second amino acid sequence by a peptidebond or a linker. The first nucleotide sequence can be linked to asecond nucleotide sequence by a phosphodiester bond or a linker. Thelinker can be a peptide or a polypeptide (for polypeptide chains) or anucleotide or a nucleotide chain (for nucleotide chains) or any chemicalmoiety (for both polypeptide and polynucleotide chains). The term“linked” is also indicated by a hyphen (-).

As used herein, the term “T cell-mediated response” refers to a responsemediated by T cells, including effector T cells (e.g., CD8⁺ cells) andhelper T cells (e.g., CD4⁺ cells). T cell mediated responses include,for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8⁺ T cells.

As used herein, the terms “inhibits” or “blocks” (e.g., referring toinhibition/blocking of binding of IL2 to IL2Rα on cells) are usedinterchangeably and encompass both partial and completeinhibition/blocking. In some embodiments, the IL2/IL2Rα fusion proteininhibits binding of IL2 to IL2Rα by at least about 50%, for example,about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, determined, e.g., asfurther described herein. In some embodiments, the IL2/IL2Rα fusionprotein inhibits binding of IL2 to IL2Rα by no more than 50%, forexample, by about 40%, 30%, 20%, 10%, 5% or 1%, determined, e.g., asfurther described herein.

As used herein, the phrase “inhibits growth of a tumor” includes anymeasurable decrease in the growth of a tumor, e.g., the inhibition ofgrowth of a tumor by at least about 10%, for example, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 99%, or 100%.

As used herein, “cancer” refers a broad group of diseases characterizedby the uncontrolled growth of abnormal cells in the body. Unregulatedcell division can result in the formation of malignant tumors or cellsthat invade neighboring tissues and can metastasize to distant parts ofthe body through the lymphatic system or bloodstream.

The terms “treat,” “treating,” and “treatment,” as used herein, refer toany type of intervention or process performed on, or administering anactive agent to, the subject with the objective of reversing,alleviating, ameliorating, inhibiting, or slowing down or preventing theprogression, development, severity or recurrence of a symptom,complication, condition or biochemical indicia associated with a diseaseor enhancing overall survival. Treatment can be of a subject having adisease or a subject who does not have a disease (e.g., forprophylaxis). When provided prophylactically, the fusion proteindisclosed herein is provided in advance of any symptom. The prophylacticadministration of the substance serves to prevent or attenuate anysubsequent symptom.

By “enhancing the efficacy” or “enhancing the immunogenicity” withregard to a fusion protein, pharmaceutical composition, or vaccine isintended improving an outcome, for example, as measured by a change in aspecific value, such as an increase or a decrease in a particularparameter of an activity of a fusion protein, pharmaceuticalcomposition, or vaccine associated with protective immunity. In oneembodiment, enhancement refers to at least a 5%, 10%, 25%, 50%, 100% orgreater than 100% increase in a particular parameter. In anotherembodiment, enhancement refers to at least a 5%, 10%, 25%, 50%, 100% orgreater than 100% decrease in a particular parameter. In one example,enhancement of the efficacy/immunogenicity of a vaccine refers to anincrease in the ability of the vaccine to inhibit or treat diseaseprogression, such as at least a 5%, 10%, 25%, 50%, 100%, or greater than100% increase in the effectiveness of the vaccine for that purpose. In afurther example, enhancement of the efficacy/immunogenicity of a vaccinerefers to an increase in the ability of the vaccine to recruit thesubject's natural defenses against cancers that have already developed,such as at least a 5%, 10%, 25%, 50%, 100%, or greater than 100%increase in the effectiveness of the fusion protein, pharmaceuticalcomposition, or vaccine for that purpose.

Similarly, by “overcoming a suppressed immune response” with regard to afusion protein, pharmaceutical composition, or vaccine is intendedimproving an outcome, for example, as measured by a change in a specificvalue, such as a return to a formerly positive value in a particularparameter of an activity of a vaccine associated with protectiveimmunity. In one embodiment, overcoming refers to at least a 5%, 10%,25%, 50%, 100% or greater than 100% increase in a particular parameter.In one example, overcoming a suppressed immune response to a fusionprotein, pharmaceutical composition, or vaccine refers to a renewedability of the fusion protein, pharmaceutical composition, or vaccine toinhibit or treat disease progression, such as at least a 5%, 10%, 25%,50%, 100%, or greater than 100% renewal in the effectiveness of thevaccine for that purpose.

A “therapeutically effective amount,” “therapeutic dose,” “dose,”“effective dose,” “effective dosage,” or “dosing amount” as used(interchangeably) herein, means a dose that achieves a therapeutic goal,as described herein. In some embodiments, a “therapeutic dose” means adose that induces an immune tolerance in a subject. In certainembodiments, a “therapeutic dose” means a dose that induces an immunetolerance in a subject within a specified time to tolerance period,e.g., within 12 weeks of administration of the first dose. A“therapeutically effective amount” of an IL2/IL2Rα fusion protein refersto the amount of the IL2/IL2Rα fusion protein sufficient to elicit adesired biological response. As will be appreciated by one of ordinaryskill in the art, the absolute amount of a particular IL2/IL2Rα fusionprotein that is effective can vary depending on such factors as thedesired biological endpoint, the IL2/IL2Rα fusion protein to bedelivered, the target cell or tissue, and the like. One of ordinaryskill in the art will further understand that an effective amount can beadministered in a single dose, or can be achieved by administration ofmultiple doses (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses). Theability of a therapeutic agent to promote disease regression or inhibitthe development or recurrence of the disease can be evaluated using avariety of methods known to the skilled practitioner, such as in humansubjects during clinical trials, in animal model systems predictive ofefficacy in humans, or by assaying the activity of the agent in in vitroassays.

“Treat,” “treatment,” or “treating,” as used herein refers to, e.g., thereduction in severity of a disease or condition; the reduction in theduration of a condition course; the amelioration or elimination of oneor more symptoms associated with a disease or condition; the provisionof beneficial effects to a subject with a disease or condition, withoutnecessarily curing the disease or condition.

By way of example, an anti-cancer agent is a drug that promotes cancerregression in a subject. In some embodiments, a therapeuticallyeffective amount of the drug promotes cancer regression to the point ofeliminating the cancer. “Promoting cancer regression” means thatadministering an effective amount of the drug, alone or in combinationwith an antineoplastic agent, results in a reduction in tumor growth orsize, necrosis of the tumor, a decrease in severity of at least onedisease symptom, an increase in frequency and duration of diseasesymptom-free periods, a prevention of impairment or disability due tothe disease affliction, or otherwise amelioration of disease symptoms inthe patient. In addition, the terms “effective” and “effectiveness” withregard to a treatment includes both pharmacological effectiveness andphysiological safety. Pharmacological effectiveness refers to theability of the drug to promote cancer regression in the patient.Physiological safety refers to the level of toxicity, or other adversephysiological effects at the cellular, organ and/or organism level(adverse effects) resulting from administration of the drug.

By way of example for the treatment of tumors, a therapeuticallyeffective amount or dosage of the drug inhibits cell growth or tumorgrowth by at least about 20%, by at least about 40%, by at least about60%, or by at least about 80% relative to untreated subjects. In someembodiments, a therapeutically effective amount or dosage of the drugcompletely inhibits cell growth or tumor growth, i.e., inhibits cellgrowth or tumor growth by 100%. The ability of a compound to inhibittumor growth can be evaluated using the assays described infra.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit cell growth, suchinhibition can be measured in vitro by assays known to the skilledpractitioner. In some embodiments described herein, tumor regression canbe observed and continue for a period of at least about 20 days, atleast about 40 days, or at least about 60 days.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions described herein canbe used to treat a subject having cancer. The term “non-human animal”includes all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, reptiles,etc.

The term “weight based” dose or dosing as referred to herein means thata dose that is administered to a patient is calculated based on theweight of the patient. For example, when a patient with 60 kg bodyweight requires 3 mg/kg of an anti-IL2 antibody, one can calculate anduse the appropriate amount of the IL2/IL2Rα fusion protein (i.e., 180mg) for administration.

The use of the term “flat dose” with regard to the methods and dosagesdescribed herein means a dose that is administered to a patient withoutregard for the weight or body surface area (BSA) of the patient. Theflat dose is therefore not provided as a mg/kg dose, but rather as anabsolute amount of the agent (e.g., the IL2/IL2Rα fusion protein). Forexample, a 60 kg person and a 100 kg person would receive the same doseof an antibody (e.g., 480 mg of an IL2/IL2Rα fusion protein).

As used herein, the terms “ug” and “uM” are used interchangeably with“μg” and “μM,” respectively.

Various aspects described herein are described in further detail in thefollowing subsections.

References made to amino acid numbering of immunoglobulins orimmunoglobulin fragments, or regions, are all based on Kabat et al.1991, Sequences of Proteins of Immunological Interest, U.S. Departmentof Public Health, Bethesda; MD. (The FcRn receptor has been isolatedfrom several mammalian species including humans. The sequences of thehuman FcRn, rat FcRn, and mouse FcRn are known (Story et al., J. Exp.Med. 180: 2377 (1994).) An Fc can comprise the CH2 and CH3 domains of animmunoglobulin with or without the hinge region of the immunoglobulin.Exemplary Fc variants are provided in WO 2004/101740 and WO 2006/074199.

7.3 Interleukin-2/Interleukin-2 Receptor Alpha Fusion Proteins

Disclosed herein is a fusion protein comprising at least two components:(a) a first polypeptide comprising an Interleukin-2 (IL2) polypeptide;and (b) a second polypeptide comprising an extracellular domain of anInterleukin-2 Receptor alpha (IL2Rα) polypeptide. In some embodiments,the extracellular domain of the IL2Rα polypeptide has at least one fewerglycosylation compared to the extracellular domain of native IL2Rα (SEQID NO:7); and/or (ii) the IL2 polypeptide has at least one fewerglycosylation compared to native IL2 (SEQ ID NO:2). In some embodiments,the fusion protein has IL2 activity.

7.3.1 Interleukin-2

In some embodiments, a fusion protein is provided which comprises afirst polypeptide comprising interleukin-2 (IL2) fused in frame to asecond polypeptide comprising or consisting of the extracellular domainof the Interleukin-2 Receptor Alpha (IL2Rα) polypeptide. In someembodiments the fusion protein comprises a first polypeptide comprisingIL2 having SEQ ID NO:2. In some embodiments, the first polypeptidecomprises an amino acid sequence at least about 60%, at least about 70%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or about 100% identical to SEQ ID NO:2.

In one embodiment, the IL2 polypeptide is a native or recombinant IL2from any vertebrate source, including mammals such as primates (e.g.,humans) and rodents (e.g., mice and rats), and domesticated oragricultural mammals unless otherwise indicated.

IL2 polypeptides useful for this disclosure are expressed in a fusionprotein. Fusion proteins described herein specifically bind human IL2R,and more specifically, a particular domain (e.g., a functional domain)within the extracellular domain of human IL2Rα. In some embodiments, thefusion protein comprising IL2 is an antagonist. In some embodiments, thefusion protein comprising IL2 binds to human IL2Rα with high affinity.

The term IL2 encompasses unprocessed IL2, as well as, any form of IL2that results from processing in the cell (i.e., the mature form of IL2).The term also encompasses naturally occurring variants and fragments ofIL2, e.g., splice variants or allelic variants, and non-naturallyoccurring variants. The amino acid sequence of an exemplary mature formof human IL2 (having the 20 amino acid signal sequence) is shown in SEQID NO:2. Unprocessed human IL2 additionally comprises an N-terminal 20amino acid signal peptide (SEQ ID NO:1), which is absent in the matureIL2 molecule. The amino acid sequence of an exemplary mature form ofmouse IL2 (having the 20 amino acid signal sequence) is shown in SEQ IDNO:4. Unprocessed mouse IL2 additionally comprises an N-terminal 20amino acid signal peptide (SEQ ID NO:3), which is absent in the matureIL2 molecule. By a “native IL2”, also termed “wild-type IL2”, is meant anaturally occurring or recombinant IL2.

Additional nucleic acid and amino acid sequences for IL2 are known. See,for example, GenBank Accession Nos: Q7JFM2 (Aotus lemurinus(Gray-bellied night monkey)); Q7JFM5 (Aotus nancymaae (Ma's nightmonkey)); P05016 (Bas taurus (Bovine)); Q29416 (Canis familiaris (Dog)(Canis lupus familiaris)); P36835 (Capra hircus (Goat)); and, P37997(Equus caballus (Horse).

Biologically active fragments and variants of IL2 are also provided.Such IL2 active variants or fragments will retain IL2 activity.Biological activity of IL2 can refer to the ability to stimulate IL2receptor bearing lymphocytes. Such activity can be measured both invitro and in vivo. IL2 is a global regulator of immune activity and theeffects seen here are the sum of such activities. For example, it isregulates survival activity (Bcl-2), induces T effector activity(IFN-gamma, Granzyme B, and Perforin), and promotes T regulatoryactivity (FoxP3). See, for example, Malek et al. (2010) Immunity33(2):153-65.

Biologically active variants of IL2 are known. See, for example, USApplication Publications 2006/0269515 and 2006/0160187 and WO 99/60128.

Biologically active fragments and variants of IL2 can be employed in thefusion proteins disclosed herein. Such a functional fragment cancomprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,50, 75, 100, 125, 150 or more continuous amino acids of SEQ ID NO:2.Alternatively, a functional variant can comprise at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity tothe sequence set forth in SEQ ID NO:2.

Active variants and fragments of polynucleotides encoding the IL2proteins are further provided. Such polynucleotide can comprise at least100, 200, 300, 400, 500, 600, 700 continuous nucleotides of polypeptideencoding SEQ ID NO:2, and continue to encode a protein having IL2activity. Alternatively, a functional polynucleotide can comprise atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to the polypeptide encoding the amino sequence setforth in SEQ ID NO:2 and continue to encode a functional IL2polypeptide.

Exemplary polypeptide sequences of IL2 are recited in Table 1, below.

TABLE 1 SEQ ID Descrip- NO tion Sequence 1 IL2 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQL (human, EHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPK un-KATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP processed)RDLISNINVIVLELKGSETTFMCEYADETATIVE FLNRWITFCQSIISTLT 2 IL2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP (human, KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE matureVLNLAQSKNFHLRPRDLISNINVIVLELKGSETT form) FMCEYADETATIVEFLNRWITFCQSIISTLT3 IL2  MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEA (mouse,QQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNL un-KLPRMLTFKFYLPKQATELKDLQCLEDELGPLRH processed)VLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDN TFECQFDDESATVVDFLRRWIAFCQSIISTSPQ 4IL2  APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMD (mouse, LQELLSRMENYRNLKLPRMLTFKFYLPKQATELK matureDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFIS form)NIRVTVVKLKGSDNTFECQFDDESATVVDFLRRW IAFCQSIISTSPQ

In some embodiments, the fusion proteins provided herein can comprise atleast one mutation within the extracellular domain of IL2Rα. In someembodiments, the IL2 polypeptide has at least one fewer glycosylationsite compared to native IL2 (SEQ ID NO:2). In some embodiments, the atleast one fewer glycosylation sites is due to one or more mutations thatremoves a glycosylation.

In other embodiments, the fusion protein comprises a mutation that is asubstitution of an amino acid having a glycosylation site with an aminoacid not having a glycosylation site. In some embodiments, the mutationremoves an O-glycosylation and/or an N-glycosylation. In one embodiment,the mutation removes an O-glycosylation, e.g., threonine at amino acid 3of SEQ ID NO: 2. In another embodiment, the mutation removes anN-glycosylation.

In some embodiments, the mutation is one or more substitutions of anamino acid of IL2 that is glycosylated with an amino acid that is notglycosylated. In some embodiments, the mutation is one or moresubstitutions of an amino acid of IL2 that allows glycosylation at anearby amino acid with an amino acid that does not allow glycosylationat the nearby amino acid.

In some embodiments, the one or more substitutions of an amino acid ofIL2 are from an alanine to an amino acid selected from the groupconsisting of arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine.

In some embodiments, the one or more substitutions of an amino acid ofIL2 are from a threonine to an amino acid selected from the groupconsisting of alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, andvaline.

In some embodiments, the one or more substitutions of an amino acid ofIL2 are from a reactive amino acid, e.g., a cysteine, to an amino acidselected from the group consisting of alanine, arginine, asparagine,aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. In some embodiments, the one or moresubstitutions are from a cysteine to a serine. In some embodiments, theone or more substitutions are from a cysteine to an alanine. In someembodiments, the one or more substitutions are from a cysteine to avaline.

In some embodiments, the one or more substitutions are at amino acid T3of IL2 compared to corresponding to SEQ ID NO:2.

In some embodiments, the one of the substitutions is at amino acid C125of SEQ ID NO:2. In a particular embodiment, the substitution at aminoacid C125 is selected from the group consisting of C125S, C125A, andC125V.

In some embodiments, the mutation is a deletion. In particularembodiments, the deletion is at amino acid A1 of SEQ ID NO:2.

The present disclosure also includes any other mutations to the IL2polypeptide. In other embodiments, the mutations also include one ormore substitutions that improve the properties of IL2, e.g., improve IL2activity, improve a half-life of IL2, improve stability, etc.

As disclosed below in this section, the mutations recited herein aremutations relative to amino acid positions of SEQ ID NO:2. According tothe present invention, any of the mutations below alone or incombination with the other disclosed mutations or any known in the artcould be used in one or more of the IL2 fusion proteins as describedherein.

In some embodiments, IL2 comprises one or more mutations disclosed inCarmenate et al., J Immunol, 200 (10) 3475-3484 (2018) and/or in U.S.Pat. No. 8,759,486: for example, at amino acid residue Q22, Q126, I129,S130, or any combination thereof, e.g., Q22V, Q126A, I129D, S130G, orany combination thereof. In some embodiments, IL2 comprises one or moremutations of L18N, Q126Y, and S130R as disclosed in U.S. Pat. No.8,759,486 B2. In some embodiments, IL2 comprises one or more mutationsof Q13Y, Q126Y, I129D, and S130R as disclosed in U.S. Pat. No. 8,759,486B2. In some embodiments, IL2 comprises one or more mutations of K35E,K35D, and K35Q as disclosed in WO 2018/091003 A1.

In some embodiments, IL2 comprises one or more mutations disclosed inEpstein et al. Blood, 101(12):4853-61] (2003) and/or in U.S. Pat. No.7,371,371: for example, at amino acid residue R38, e.g., R38W. In someembodiments, IL2 comprises the mutation of R38W and one or moremutations outside of amino acid positions 22 to 58 of IL2 as disclosedin U.S. Pat. No. 7,371,371 B2.

In some embodiments, IL2 comprises one or more mutations disclosed inWittrup et al. J Immunother. 32(9):887-94 (2009) and/or in U.S. Pat. No.8,906,356: for example, amino acid residue 91, 126, or both, e.g., V91R,Q126T, or both. In some embodiments, IL2 comprises the mutation of E15Was disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) andalso in U.S. Pat. No. 8,906,356. In some embodiments, IL2 comprises oneor both mutations of N88R and V91R as disclosed in Wittrup et al. JImmunother. 32(9):887-94 (2009) and also in U.S. Pat. No. 8,906,356. Insome embodiments, IL2 comprises the mutation of Q126T or Q126I asdisclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) and/or inU.S. Pat. No. 8,906,356.

In some embodiments, IL2 comprises one or more mutations disclosed inU.S. Pat. No. 8,906,356 B2: for example at amino acid 69, 74, 91, 126,or any combination thereof. In some embodiments, the mutation is V91R,Q126T, Q126L, Q127T, or any combination thereof as disclosed in U.S.Pat. No. 8,906,356 B2.

In some embodiments, IL2 comprises one or more mutations disclosed inWittrup et al. J Immunother. 32(9):887-94 (2009) and/or in U.S. Pat. No.7,569,215 B2: for example, at amino acid residue E15, N30, E68, V69,N71, S75, N90, or any combination thereof, e.g., N30S, E68D, V69A, N71A,S75P, N90H, or any combination thereof. In some embodiments, IL2comprises the mutation of E15W as disclosed in Wittrup et al.Biochemistry, Vol. 44, No. 31 (2005). In some embodiments, the mutationis V69A as disclosed in U.S. Pat. No. 7,569,215 B2.

In some embodiments, IL2 comprises one or more mutations disclosed inWittrup et al. J Immunother. 32(9):887-94 (2009) and/or in U.S. Pat. No.7,951,360 B2: for example, at amino acid residue N29, Y31, K35, T37,K48, V69, N71, N88, or any combination thereof, e.g., N29S, Y31H, K35R,T37A, K48E, V69A, N71R, N88D, or any combination thereof. In someembodiments, IL2 comprises the mutation of E15W as disclosed in Wittrupet al. Biochemistry, Vol. 44, No. 31 (2005).

In some embodiments, IL2 comprises one or more mutations disclosed inWittrup et al. J Immunother. 32(9):887-94 (2009) and/or in U.S. Pat. No.8,349,311 B2: for example, at amino acid 69, 74, 128, or any combinationthereof, e.g., V69A, I128P, or any combination thereof.

In some embodiments, IL2 comprises one or more mutations disclosed inWittrup et al. J Immunother. 32(9):887-94 (2009): for example, at aminoacid residue S4, T10, Q11, V69, N88, T133, or any combination thereof,e.g., S4P, T10A, Q11R, V69A, N88D, T133A, or any combination thereof.

In some embodiments, IL2 comprises one or more mutations disclosed inWittrup et al. J Immunother. 32(9):887-94 (2009): for example, at aminoacid residue N30, V69, I128, or any combination thereof, e.g., N30S,V69A, I128T, or any combination thereof.

In some embodiments, IL2 comprises one or more mutations disclosed inWittrup et al. J Immunother. 32(9):887-94 (2009): for example, at aminoacid residue K8, Q13, N26, N30, K35, T37, V69, or any combinationthereof, e.g., K8R, Q13R, N26D, N30T, K35R, T37R, V69A, or anycombination thereof.

In some embodiments, IL2 comprises one or more mutations disclosed inShanafelt et al., Nat Biotechnol., 18(11):1197-202 (2000) for example,at amino acid residue N88, e.g., N88R.

In some embodiments, IL2 comprises one or more mutations disclosed inU.S. Pat. No. 9,616,105 B2: for example, amino acids 20, 88, 126, or anycombination thereof, e.g., N88R, N88G, or N88I. In some embodiments, IL2comprises a mutation of N88R, N88G, or N88I as disclosed in U.S. Pat.No. 9,616,105 B2. In some embodiments, IL2 comprises a mutation of D20H,D20I, or D20Y as disclosed in U.S. Pat. No. 9,616,105 B2. In someembodiments, IL2 comprises the mutation of Q126L as disclosed in U.S.Pat. No. 9,616,105 B2.

In some embodiments, IL2 comprises one or more mutations disclosed in US2018/0125941 A1: for example, D20H, N88I, N88G, N88R, Q126L, Q126F, orany combination thereof. In some embodiments, IL2 comprises one or moremutations of T3A, N88G, N88R, D20H, C125S, Q126L, and Q126F as disclosedin US 2018/0037624 A1.

In some embodiments, IL2 comprises one or more mutations disclosed in US2017/0327555 A1: for example, at amino acid residue N88, D20, C125,Q126, or any combination thereof, e.g., N88G, N88R, D20H, C125S, Q126L,Q126F, or any combination thereof.

In some embodiments, IL2 comprises one or more mutations disclosed in WO2016/025385 A1: for example, at amino acid residue D109, C125, or both,e.g., D109C, C125S, or both. In some embodiments, IL2 comprises one ormore mutations disclosed in WO 2016/025385 A1: for example; at aminoacid residue D20, N88, Q126, C125, Q126, or any combination thereof,e.g., D20H, N88I, N88G, N88R, Q126L, C125S, Q126F, or any combinationthereof.

In some embodiments, IL2 comprises one or more mutations disclosed in WO2016/164937 A1: for example, at amino acid residue L12, Q13, E15, H16,L19, D20, M23, D84, S87, N88, V91, E95, or any combination thereof,e.g., L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G,H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G,L19N, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, D84A,D84E, D84G, D841, D84M, D84Q, D84R, D84S, D84T, S87E, N88A, N88D, N88E,N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91S, E95G,or any combination thereof.

In some embodiments, IL2 comprises one or more mutations disclosed inU.S. Pat. No. 9,932,380 B2 or U.S. Pat. No. 9,580,486: for example, atamino acid residue V91, e.g., V91K. In some embodiments, IL2 furthercomprises a mutation of C125A or C125S. In some embodiments, IL2 furthercomprises a mutation at T3. In some embodiments, the mutation at T3 isone of T3A or T3N. In some embodiments, IL2 comprises a mutation at S5.In some embodiments, the mutation is S5T.

In some embodiments, IL2 comprises one or more mutations disclosed inU.S. Pat. No. 9,732,134 B2: for example, E15, H16, Q22, D84, N88, E95,or any combination thereof.

In some embodiments, IL2 comprises one or more mutations disclosed in US2015/0218260 A1: for example, N88D. In some embodiments, IL2 comprises amutation disclosed in U.S. Pat. No. 9,266,938 B2: for example, at aminoacid 42, 45, 72, or any combination thereof, e.g., L72G, L72A, L72S,L72T, L72Q, L72E, L72N, L72D, L72R, or L72K. In some embodiments, IL2comprises a mutation of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D,F42R, and F42K. In some embodiments, IL2 comprises a mutation of Y45A,Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K.

In some embodiments, IL2 comprises one to four mutations: the firstmutation of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, orL72K, the second mutation of F42A, F42G, F42S, F42T, F42Q, F42E, F42N,F42D, F42R, or F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D,Y45R, or Y45K, the third mutation of T3A, T3G, T3Q, T3E, T3N, T3D, T3R,T3K, or T3P, and/or the fourth mutation of C125A, C125S, C125T or C125V.The mutations listed herein or disclosed in the patents, patentpublications or any other references cited herein are incorporatedherein by reference in their entireties.

7.3.2 Interleukin-2 Receptor Alpha

The fusion protein comprises a second polypeptide comprising theextracellular domain of the Interleukin-2 Receptor Alpha (IL2Rα). Insome embodiments, the extracellular domain of IL2Rα comprises the aminoacid sequence set forth as SEQ ID NO:7. In some embodiments, the secondpolypeptide comprises an amino acid sequence at least about 60%, atleast about 70%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or about 100% identical to SEQ IDNO:7.

The term “CD25” or “IL2 receptor a,” “IL2Rα,” “IL2Ra,” “IL2-Rα,” and“IL2-Ra” as used herein, refers to any native or recombinant IL2Rα fromany vertebrate source, including mammals such as primates (e.g., humans)and rodents (e.g., mice and rats) and domesticated or agriculturalmammals unless otherwise indicated. The term also encompasses naturallyoccurring variants of IL2Rα, e.g., splice variants or allelic variants,or non-naturally occurring variants. Human IL2 exerts its biologicaleffects via signaling through its receptor system, IL2R. IL2 and itsreceptor (IL2R) are required for T-cell proliferation and otherfundamental functions which are crucial of the immune response. IL2Rconsists of 3 non-covalently linked type I transmembrane proteins whichare the alpha (p55), beta (p75), and gamma (p65) chains. The human IL2Ralpha chain contains an extracellular domain of 219 amino acids, atransmembrane domain of 19 amino acids, and an intracellular domain of13 amino acids. The secreted extracellular domain of IL2R alpha (IL2Rα)can be employed in the fusion proteins describe herein.

The amino acid sequence of an exemplary mature form of human IL2Rα isshown in SEQ ID NO:6. Unprocessed human IL2Rα is shown in SEQ ID NO:5.The extracellular domain of SEQ ID NO:5 and/or SEQ ID NO:6 is set forthin SEQ ID NO:7. The amino acid sequence of an exemplary mature form ofmouse IL2Rα is shown in SEQ ID NO:9. Unprocessed mouse IL2Rα is shown inSEQ ID NO:8. The extracellular domain of SEQ ID NO:8 and/or SEQ ID NO:9is set forth in SEQ ID NO:10. By a “native IL2Rα”, also termed“wild-type IL2Rα”, is meant a naturally occurring or recombinant IL2Rα.

Nucleic acid and amino acid sequences for IL2Rα are known. See, forexample, GenBank Accession Nos: NP_001030597.1 (P. troglodytes);NP_001028089.1 (M. mulatta); NM_001003211.1 (C. lupus); NP_776783.1 (B.taurus); NP_032393.3 (M. musculus); and, NP_037295.1 (R. norvegicus).

The extracellular domain of IL2Rα as used herein means a functionalIL2Rα extracellular domain in its normal role in binding to IL2, unlessotherwise specified. The term a IL2Rα EC domain includes a functionalfragment, variant, analog, or derivative thereof that retains thefunction of full-length wild-type IL2Rα EC in IL2 binding. The IL2Rα ECdomain can be the human, porcine, canine, rat, or murine IL2Rα ECdomain. The phrase “biological activity of the IL2Rα EC domain” refersto one or more of the biological activities of EC domain of IL2Rα,including but not limited to, the ability to enhance intracellularsignaling in IL2 receptor responsive cells. Non-limiting examples ofbiologically active fragments and variants of the IL2Rα EC domain aredisclosed, for example, in Robb et al., Proc. Natl. Acad. Sci. USA,85:5654-5658, 1988. In some embodiments, the biologically activefragments and variants of the IL2Rα EC domain disclosed herein compriseat least one fewer glycosylation compared to the extracellular domain ofnative IL2Rα.

Biologically active fragments and variants of the extracellular domainof IL2Rα can be employed in the fusion proteins disclosed herein. Such afunctional fragment can comprise at least 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 215 or greatercontinuous amino acids of the extracellular domain of any one of SEQ IDNO:7. Alternatively, a functional variant can comprise at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the sequence set forth in SEQ ID NO:7.

Active variants and fragments of polynucleotides encoding theextracellular domain of IL2Rα are further provided. Such polynucleotidecan comprise at least 100, 200, 300, 400, 500, 600 or greater continuousnucleotides of polypeptide encoding SEQ ID NO:7 and continue to encode aprotein having the extracellular domain activity of IL2Rα.Alternatively, a functional polynucleotide can comprise at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the polypeptide encoding the amino sequence set forth in SEQID NO:7 and continue to encode a protein having the extracellular domainactivity of IL2Rα.

Polypeptide sequences of IL2Rα are recited in Table 2.

TABLE 2 SEQ ID Descrip- NO tion Sequence  5 IL2RαMDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHAT (human,FKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGN un-SSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERK processedTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY form)HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKT RWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAG CVFLLISVLLLSGLTWQRRQRKSRRTI  6 IL2RαELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFR (human,RIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTT matureKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCR form)EPPPWENEATERIYHFVVGQMVYYQCVQGYRALHR GPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATME TSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI  7 IL2Rα ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFR (human,RIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTT matureKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCR form ofEPPPWENEATERIYHFVVGQMVYYQCVQGYRALHR IL2RαGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGE extracel-EKPQASPEGRPESETSCLVTTTDFQIQTEMAATME lular TSIFTTEYQ domain)  8 IL2RαMEPRLLMLGFLSLTIVPSCRAELCLYDPPEVPNAT (mouse,FKALSYKNGTILNCECKRGFRRLKELVYMRCLGNS un-WSSNCQCTSNSHDKSRKQVTAQLEHQKEQQTTTDM processedQKPTQSMHQENLTGHCREPPPWKHEDSKRIYHFVE form)GQSVHYECIPGYKALQRGPAISICKMKCGKTGWTQ PQLTCVDEREHHRFLASEESQGSRNSSPESETSCPITTTDFPQPTETTAMTETFVLTMEYKVAVASCLFL LISILLLSGLTWQHRWRKSRRTI  9 IL2RαELCLYDPPEVPNATFKALSYKNGTILNCECKRGFR (mouse,RLKELVYMRCLGNSWSSNCQCTSNSHDKSRKQVTA matureQLEHQKEQQTTTDMQKPTQSMHQENLTGHCREPPP form)WKHEDSKRIYHFVEGQSVHYECIPGYKALQRGPAI SICKMKCGKTGWTQPQLTCVDEREHHRFLASEESQGSRNSSPESETSCPITTTDFPQPTETTAMTETFVL TMEYKVAVASCLFLLISILLLSGLTWQHRWRKSRRTI 10 IL2Rα ELCLYDPPEVPNATFKALSYKNGTILNCECKRGFR (mouse,RLKELVYMRCLGNSWSSNCQCTSNILRASHDKSRK matureQVTAQLEHQKEQQTTTDMQKPTQSMHQENLTGHCR form ofEPPPWKHEDSKRIYHFVEGQSVHYECIPGYKALQR IL2RαGPAISICKMKCGKTGWTQPQLTCVDEREHHRFLAS extracel-EESQGSRNSSPESETSCPITTTDFPQPTETTAMTE lular TFVLTMEYK domain) 11 IL2RαELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFR (human,RIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTT matureKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCR form ofEPPPWENEATERIYHFVVGQMVYYQCVQGYRALHR IL2Rα GPAESVCKMTHGKTRWTQPQLICTGEextracel- lular domain)- full- truncated 12 IL2RαELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFR (human,RIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTT matureKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCR form ofEPPPWENEATERIYHFVVGQMVYYQCVQGYRALHR IL2RαGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGE extracel- EKPQASPEGRPESETS lulardomain)- half- truncated

In some embodiments, the fusion proteins provided herein can comprise atleast one mutation within the EC domain of IL2Rα.

In some embodiments, the EC domain of the IL2Rα polypeptide has at leastone fewer glycosylation, at least two fewer glycosylations, at leastthree fewer glycosylations, at least four fewer glycosylations, at leastfive fewer glycosylations, at least six fewer glycosylations, at leastseven fewer glycosylations, at least eight fewer glycosylations, or atleast nine fewer glycosylations compared to the extracellular domain ofnative IL2Rα (SEQ ID NO:7).

In some embodiments, the EC domain of the IL2Rα polypeptide having atleast one fewer glycosylation comprises a mutation that removes aglycosylation. In other embodiments, the fusion protein comprises amutation that is a substitution of an amino acid having a glycosylationsite with an amino acid not having a glycosylation site. In someembodiments, the mutation removes an O-glycosylation and/or anN-glycosylation. In one embodiment, the mutation removes anO-glycosylation. In another embodiment, the mutation removes anN-glycosylation.

In some embodiments, the mutation in the fusion protein comprises adeletion of the C-terminal end of IL2Rα. In some embodiments, themutation is a deletion of amino acids 167 to 219 of SEQ ID NO:7. In someembodiments, the mutation is a deletion of amino acids 168 to 219 of SEQID NO:7. In some embodiments, the mutation is a deletion of amino acids169 to 219 of SEQ ID NO:7. In some embodiments, the mutation is adeletion of amino acids 170 to 219 of SEQ ID NO:7. In some embodiments,the mutation is a deletion of amino acids 171 to 219 of SEQ ID NO:7. Insome embodiments, the mutation is a deletion of amino acids 172 to 219of SEQ ID NO:7. In some embodiments, the mutation is a deletion of aminoacids 173 to 219 of SEQ ID NO:7. In some embodiments, the mutation is adeletion of amino acids 174 to 219 of SEQ ID NO:7. In some embodiments,the mutation is a deletion of amino acids 175 to 219 of SEQ ID NO:7. Insome embodiments, the mutation is a deletion of amino acids 176 to 219of SEQ ID NO:7. In some embodiments, the mutation is a deletion of aminoacids 177 to 219 of SEQ ID NO:7. In some embodiments, the mutation is adeletion of amino acids 178 to 219 of SEQ ID NO:7. In some embodiments,the mutation is a deletion of amino acids 179 to 219 of SEQ ID NO:7. Insome embodiments, the mutation is a deletion of amino acids 180 to 219of SEQ ID NO:7. In some embodiments, the mutation is a deletion of aminoacids 181 to 219 of SEQ ID NO:7. In some embodiments, the mutation is adeletion of amino acids 182 to 219 of SEQ ID NO:7. In some embodiments,the mutation is a deletion of amino acids 183 to 219 of SEQ ID NO:7. Insome embodiments, the mutation is a deletion of amino acids 184 to 219of SEQ ID NO:7. In some embodiments, the mutation is a deletion of aminoacids 185 to 219 of SEQ ID NO:7. In some embodiments, the mutation is adeletion of amino acids 186 to 219 of SEQ ID NO:7. In some embodiments,the mutation is a deletion of amino acids 187 to 219 of SEQ ID NO:7. Insome embodiments, the mutation is a deletion of amino acids 188 to 219of SEQ ID NO:7. In some embodiments, the mutation is a deletion of aminoacids 189 to 219 of SEQ ID NO:7. In some embodiments, the mutation is adeletion of amino acids 190 to 219 of SEQ ID NO:7. In some embodiments,the mutation is a deletion of amino acids 191 to 219 of SEQ ID NO:7. Insome embodiments, the mutation is a deletion of amino acids 192 to 219of SEQ ID NO:7.

In some embodiments, the mutation is a deletion of amino acids from 167,168, 169 or 171 through 192 to 219, corresponding to SEQ ID NO:7. Insome embodiments, the mutation does not include a deletion of 170 to219, corresponding to SEQ ID NO:7.

In some embodiments, the second polypeptide comprises, consistsessentially of, or consists of an amino acid sequence at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or about 100% identical to the amino acid sequence set forth in SEQID NO:11. In other embodiments, the second polypeptide comprises,consists essentially of, or consists of SEQ ID NO: 11 and +1, +2, +3,+4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18,+19, +20, +21, +22, +23, +24, or +25 amino acids. In some embodiments,the second polypeptide comprises, consists essentially of, or consistsof SEQ ID NO: 11 with no more than 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. Insome embodiments, the second polypeptide comprises, consists essentiallyof, or consists of an amino acid sequence at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or about100% identical to the amino acid sequence set forth in SEQ ID NO:12.

In some embodiments, the fusion protein comprises one or more mutations.In some embodiments, the one or more mutations are one or moresubstitutions of an amino acid of IL2Rα that is glycosylated with anamino acid that is not glycosylated.

In some embodiments, the one or more substitutions amino acids of IL2Rαare at amino acid N49, amino acid N68, amino acid T74, amino acid T85,amino acid T197, amino acid T203, amino acid T208, and amino acid T216,or any combination thereof, wherein the amino acid locations correspondto SEQ ID NO:7.

In some embodiments, the one or more substitutions are from asparagineto another amino acid. In some embodiments, the one or moresubstitutions is from asparagine to an amino acid selected from thegroup consisting of alanine, threonine, serine, arginine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, andvaline.

In some embodiments, the one or more substitutions are from threonine toanother amino acid. In some embodiments, the one or more substitutionsis from threonine to an amino acid selected from the group consisting ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, tryptophan, tyrosine, andvaline.

In some embodiments, the substitution is amino acid N49 of SEQ ID NO:7.In some embodiments, amino acid N49 of SEQ ID NO:7 is mutated to anamino acid selected from the group consisting of alanine, threonine,serine, arginine, aspartic acid, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, tryptophan, tyrosine, and valine.

In some embodiments, the substitution is amino acid N68 of SEQ ID NO:7.In some embodiments, amino acid N68 of SEQ ID NO:7 is mutated to anamino acid selected from the group consisting of alanine, threonine,serine, arginine, aspartic acid, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, tryptophan, tyrosine, and valine.

In some embodiments, the substitution is amino acid T74 of SEQ ID NO:7.In some embodiments, amino acid T74 of SEQ ID NO:7 is mutated to anamino acid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine, and valine.

In some embodiments, the substitution is amino acid T85 of SEQ ID NO:7.In some embodiments, amino acid T85 of SEQ ID NO:7 is mutated to anamino acid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine, and valine.

In some embodiments, the substitution is amino acid T197 of SEQ ID NO:7.In some embodiments, amino acid T197 of SEQ ID NO:7 is mutated to anamino acid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine, and valine.

In some embodiments, the substitution is amino acid T203 of SEQ ID NO:7.In some embodiments, amino acid T203 of SEQ ID NO:7 is mutated to anamino acid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine, and valine.

In some embodiments, the substitution is amino acid T208 of SEQ ID NO:7.In some embodiments, amino acid T208 of SEQ ID NO:7 is mutated to anamino acid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine, and valine.

In some embodiments, the substitution is amino acid T216 of SEQ ID NO:7.In some embodiments, amino acid T216 of SEQ ID NO:7 is mutated to anamino acid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine, and valine.

In some embodiments, the fusion protein comprises one or more mutations.In some embodiments, the one or more mutations is one or moresubstitutions of an amino acid of IL2Rα that allows glycosylation at anearby amino acid with an amino acid that does not allow glycosylationat the nearby amino acid.

In some embodiments, the substitution is at amino acid S50, amino acidS51, amino acid T69, amino acid T70, amino acid C192, or any combinationthereof, wherein the amino acid locations correspond to SEQ ID NO:7.

In some embodiments, the substitution is amino acid S50 corresponding toSEQ ID NO:7. In some embodiments, amino acid S50 corresponding to SEQ IDNO:7 is mutated to proline.

In some embodiments, the substitution is amino acid S51 corresponding toSEQ ID NO:7. In some embodiments, amino acid S51 corresponding to SEQ IDNO:7 is mutated to an amino acid selected from the group consisting ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, tryptophan, tyrosine, andvaline.

In some embodiments, the substitution is amino acid T69 corresponding toSEQ ID NO:7. In some embodiments, amino acid T69 corresponding to SEQ IDNO:7 is mutated to proline.

In some embodiments, the substitution is amino acid T70 corresponding toSEQ ID NO:7. In some embodiments, amino acid T70 corresponding to SEQ IDNO:7 is mutated to an amino acid selected from the group consisting ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, andvaline.

In some embodiments, the substitution is amino acid C192 correspondingto SEQ ID NO:7. In some embodiments, amino acid C192 corresponding toSEQ ID NO:7 is mutated to an amino acid selected from the groupconsisting of alanine, arginine, asparagine, aspartic acid, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine.

7.3.3 Linker

The fusion protein of the present disclosure can further comprise alinker. In some embodiments, the linker can link the first polypeptideto the second polypeptide from N-terminus to C-terminus, e.g.,N-IL2-linker-IL2Rα EC-C. In other embodiments, the linker can link thesecond polypeptide to the first polypeptide from N-terminus toC-terminus, e.g., N-IL2Rα EC-linker-IL2-C.

In one embodiment, the IL2/IL2Rα fusion protein comprises a linkersequence located between the IL2 polypeptide and the IL2Rα polypeptide.The linker can be of any length and can comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 50, or 60 or more amino acids. In otherembodiments, a linker useful for the present disclosure has at least oneamino acid and less than 100 amino acids, less than 90 amino acids, lessthan 80 amino acids, less than 70 amino acids, less than 60 amino acids,less than 50 amino acids, less than 40 amino acids, less than 30 aminoacids, less than 20 amino acids, less than 19 amino acids, less than 18amino acids, less than 17 amino acids, less than 16 amino acids, lessthan 15 amino acids, less than 14 amino acids, less than 13 amino acids,or less than 12 amino acids. In one embodiment, the linker sequencecomprises glycine amino acid residues. In other instances, the linkersequence comprises a combination of glycine and serine amino acidresidues.

In some embodiments, the fusion protein comprises a linker fused inframe between the first polypeptide and the second polypeptide. In someembodiments, the fusion protein comprises a linker is a glycine/serinelinker. Such glycine/serine linkers can comprises any combination of theamino acid residues, including, but not limited to, the peptide GGGS(SEQ ID NO: 174) or GGGGS (SEQ ID NO: 72) or repeats of the same,including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of these givenpeptides. The glycine/serine linkers disclosed herein comprises an aminoacid sequence of (GS)_(n), (GGS)_(n), (GGGS)_(n), (GGGGS)_(n), or(GGGGS)_(n), wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or10. In a particular embodiment, the linker sequence comprisesGGGSGGGSGGGS (SEQ ID NO:71) (also noted as (Gly₃Ser)₃). In anotherembodiment, the linker sequence comprises GGGSGGGSGGGSGGGS (SEQ ID NO:74) (also noted as (Gly₃Ser)₄). In other embodiments, the linkersequence comprises one of (Gly₃Ser)₅ (GGGSGGGSGGGSGGGSGGGS) (SEQ ID NO:75), (Gly₃Ser)₆ (GGGSGGGSGGGSGGGSGGGSGGGS) (SEQ ID NO: 76), or(Gly₃Ser)₇ (GGGSGGGSGGGSGGGSGGGSGGGSGGGS) (SEQ ID NO: 77). In otherembodiments, the linker sequence comprises (Gly₄Ser)₃ (GGGGSGGGGSGGGGS)as set forth in SEQ ID NO: 78. In additional embodiments, the linkersequence comprises GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 79) (also noted as(Gly₄Ser)₄); GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 80) (also noted as(Gly₄Ser)₅); (Gly₄Ser)₂ (GGGGSGGGGS) (SEQ ID NO: 81), (Gly₄Ser)₁ (GGGGS)(SEQ ID NO: 82), (Gly₄Ser)₆ (GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:83); (Gly₄Ser)₇ (GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 97);or (Gly₄Ser)₅ (GGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 84).

7.3.4 Heterologous Moiety

The fusion protein of the present disclosure can further comprise anadditional element, e.g., heterologous moiety. Such elements can aid inthe expression of the fusion protein, aid in the secretion of the fusionprotein, improve the stability of the fusion protein, allow for moreefficient purification of the protein, and/or modulate the activity ofthe fusion protein. In some embodiment, the heterologous moiety is apolypeptide moiety. In other embodiments, the heterologous moiety is anon-polypeptide moiety.

In some embodiments, the fusion protein comprises a heterologous moietyfused to the first polypeptide. In some embodiments, the fusion proteincomprises a heterologous moiety fused to the second polypeptide. In someembodiments, the fusion protein comprises a heterologous moiety fused tothe first polypeptide and the second polypeptide.

In some embodiments, the fusion proteins disclosed herein comprise oneor more additional heterologous moieties. In some embodiments, theheterologous moieties are half-life extending moieties. In someembodiments, the heterologous moiety comprises albumin, animmunoglobulin constant region or a portion thereof, animmunoglobulin-binding polypeptide, an immunoglobulin G (IgG),albumin-binding polypeptide (ABP), a PASylation moiety, a HESylationmoiety, XTEN, a PEGylation moiety, an Fc region, and any combinationthereof.

1) Immunoglobulin Constant Region or Portion Thereof

An immunoglobulin constant region is comprised of domains denoted CH(constant heavy) domains (CH1, CH2, etc.). Depending on the isotype,(i.e. IgG, IgM, IgA IgD, or IgE), the constant region can be comprisedof three or four CH domains. Some isotypes (e.g. IgG) constant regionsalso contain a hinge region. See Janeway et al. 2001, Immunobiology,Garland Publishing, N.Y., N.Y.

An immunoglobulin constant region or a portion thereof for producing thefusion protein of the present disclosure may be obtained from a numberof different sources. In preferred embodiments, An immunoglobulinconstant region or a portion thereof is derived from a humanimmunoglobulin. It is understood, however, that the immunoglobulinconstant region or a portion thereof may be derived from animmunoglobulin of another mammalian species, including for example, arodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate(e.g. chimpanzee, macaque) species. Moreover, the immunoglobulinconstant region or a portion thereof may be derived from anyimmunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and anyimmunoglobulin isotype, including IgG1, IgG2, IgG3 and IgG4. In oneembodiment, the human isotype IgG1 is used.

A variety of the immunoglobulin constant region gene sequences (e.g.human constant region gene sequences) are available in the form ofpublicly accessible deposits. Constant region domains sequence can beselected having a particular effector function (or lacking a particulareffector function) or with a particular modification to reduceimmunogenicity. Many sequences of antibodies and antibody-encoding geneshave been published and suitable Ig constant region sequences (e.g.hinge, CH2, and/or CH3 sequences, or portions thereof) can be derivedfrom these sequences using art recognized techniques. The geneticmaterial obtained using any of the foregoing methods may then be alteredor synthesized to obtain polypeptides of the present disclosure. It willfurther be appreciated that the scope of this disclosure encompassesalleles, variants and mutations of constant region DNA sequences.

The sequences of the immunoglobulin constant region or a portion thereofcan be cloned, e.g., using the polymerase chain reaction and primerswhich are selected to amplify the domain of interest. To clone asequence of the immunoglobulin constant region or a portion thereof froman antibody, mRNA can be isolated from hybridoma, spleen, or lymphcells, reverse transcribed into DNA, and antibody genes amplified byPCR. PCR amplification methods are described in detail in U.S. Pat. Nos.4,683,195; 4,683,202; 4,800,159; 4,965,188; and in, e.g., “PCRProtocols: A Guide to Methods and Applications” Innis et al. eds.,Academic Press, San Diego, Calif. (1990); Ho et al. 1989. Gene 77:51;Horton et al. 1993. Methods Enzymol. 217:270). PCR may be initiated byconsensus constant region primers or by more specific primers based onthe published heavy and light chain DNA and amino acid sequences. Asdiscussed above, PCR also may be used to isolate DNA clones encoding theantibody light and heavy chains. In this case the libraries may bescreened by consensus primers or larger homologous probes, such as mouseconstant region probes. Numerous primer sets suitable for amplificationof antibody genes are known in the art (e.g., 5′ primers based on theN-terminal sequence of purified antibodies (Benhar and Pastan. 1994.Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti,F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences(Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250). Thecloning of antibody sequences is further described in Newman et al.,U.S. Pat. No. 5,658,570, filed Jan. 25, 1995.

An immunoglobulin constant region used herein can include all domainsand the hinge region or portions thereof. In one embodiment, theimmunoglobulin constant region or a portion thereof comprises CH2domain, CH3 domain, and a hinge region, i.e., an Fc region or an FcRnbinding partner.

As used herein, the term “Fc region” is defined as the portion of apolypeptide which corresponds to the Fc region of native immunoglobulin,i.e., as formed by the dimeric association of the respective Fc domainsof its two heavy chains. A native Fc region forms a homodimer withanother Fc region.

In one embodiment, the “Fc region” refers to the portion of a singleimmunoglobulin heavy chain beginning in the hinge region just upstreamof the papain cleavage site (i.e. residue 216 in IgG, taking the firstresidue of heavy chain constant region to be 114) and ending at theC-terminus of the antibody. Accordingly, a complete Fc domain comprisesat least a hinge domain, a CH2 domain, and a CH3 domain.

The Fc region of an immunoglobulin constant region, depending on theimmunoglobulin isotype can include the CH2, CH3, and CH4 domains, aswell as the hinge region. Fusion proteins comprising an Fc region of animmunoglobulin bestow several desirable properties on a fusion proteinincluding increased stability, increased serum half-life (see Capon etal., 1989, Nature 337:525) as well as binding to Fc receptors such asthe neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726,6,030,613; WO 03/077834; US2003-0235536A1).

In some embodiments, the “Fc region” includes an amino acid sequence ofan Fc domain or derived from an Fc domain. In certain embodiments, an Fcregion comprises at least one of: a hinge (e.g., upper, middle, and/orlower hinge region) domain (about amino acids 216-230 of an antibody Fcregion according to EU numbering), a CH2 domain (about amino acids231-340 of an antibody Fc region according to EU numbering), a CH3domain (about amino acids 341-438 of an antibody Fc region according toEU numbering), a CH4 domain, or a variant, portion, or fragment thereof.In other embodiments, an Fc region comprises a complete Fc domain (i.e.,a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, anFc region comprises, consists essentially of, or consists of a hingedomain (or a portion thereof) fused to a CH3 domain (or a portionthereof), a hinge domain (or a portion thereof) fused to a CH2 domain(or a portion thereof), a CH2 domain (or a portion thereof) fused to aCH3 domain (or a portion thereof), a CH2 domain (or a portion thereof)fused to both a hinge domain (or a portion thereof) and a CH3 domain (ora portion thereof). In still other embodiments, an Fc region lacks atleast a portion of a CH2 domain (e.g., all or part of a CH2 domain). Ina particular embodiment, an Fc region comprises or consists of aminoacids corresponding to EU numbers 221 to 447.

The Fc domains denoted as F, F1, or F2 herein may be obtained from anumber of different sources. In one embodiment, an Fc region of thepolypeptide is derived from a human immunoglobulin. It is understood,however, that an Fc region may be derived from an immunoglobulin ofanother mammalian species, including for example, a rodent (e.g. amouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee,macaque) species. Moreover, the polypeptide of the Fc domains orportions thereof may be derived from any immunoglobulin class, includingIgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype, includingIgG1, IgG2, IgG3 and IgG4. In another embodiment, the human isotype IgG1is used.

In certain embodiments, the Fc variant confers a change in at least oneeffector function imparted by an Fc region comprising said wild-type Fcdomain (e.g., an improvement or reduction in the ability of the Fcregion to bind to Fc receptors (e.g. FcγRI, FcγRII, or FcγRIII) orcomplement proteins (e.g. C1q), or to trigger antibody-dependentcytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity(CDCC)). In other embodiments, the Fc variant provides an engineeredcysteine residue.

The Fc regions of the disclosure may employ art-recognized Fc variantswhich are known to impart a change (e.g., an enhancement or reduction)in effector function and/or FcR binding. Specifically, a bindingmolecule of the disclosure may include, for example, a change (e.g., asubstitution) at one or more of the amino acid positions disclosed inInternational PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1,WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2,WO04/099249A2, WO005/040217A2, WO004/044859, WO05/070963A1,WO05/077981A2, WO05/092925A2, WO05/123780A2, WO06/019447A1,WO06/047350A2, and WO06/085967A2; US Patent Publication Nos.US2007/0231329, US2007/0231329, US2007/0237765, US2007/0237766,US2007/0237767, US2007/0243188, US20070248603, US20070286859,US20080057056; or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250;5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375;6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784;7,404,956, and 7,317,091. In one embodiment, the specific change (e.g.,the specific substitution of one or more amino acids disclosed in theart) may be made at one or more of the disclosed amino acid positions.In another embodiment, a different change at one or more of thedisclosed amino acid positions (e.g., the different substitution of oneor more amino acid position disclosed in the art) may be made.

The Fc region of IgG can be modified according to well recognizedprocedures such as site directed mutagenesis and the like to yieldmodified IgG or Fc fragments or portions thereof that will be bound byFc receptors. Such modifications include modifications remote from theFc receptor contact sites as well as modifications within the contactsites that preserve or even enhance binding to the Fc receptors. Forexample, the following single amino acid residues in human IgG1 Fc (Fcγ1) can be substituted without significant loss of Fc binding affinityfor Fc receptors: P238A, S239A, K246A, K248A, D249A, M252A, T256A,E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A,Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A,E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A,L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A,A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A,Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A,Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A,E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A,R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A,K439A, S440A, S444A, and K447A, where for example P238A represents wildtype proline substituted by alanine at position number 238. As anexample, a specific embodiment incorporates the N297A mutation, removinga highly conserved N-glycosylation site. In addition to alanine otheramino acids may be substituted for the wild type amino acids at thepositions specified above. Mutations may be introduced into Fc givingrise to more than one hundred Fc regions distinct from the native Fc.Additionally, combinations of two, three, or more of these individualmutations may be introduced together, giving rise to hundreds more Fcregions. Moreover, one of the Fc region of a construct of the disclosuremay be mutated and the other Fc region of the construct not mutated atall, or they both may be mutated but with different mutations.

Certain of the above mutations may confer new functionality upon the Fcregion. For example, one embodiment incorporates N297A, removing ahighly conserved N-glycosylation site. The effect of this mutation is toreduce immunogenicity, thereby enhancing circulating half-life of the Fcregion, and to render the Fc region incapable of binding to FcγRI,FcγRIIA, FcγRIIB, and FcγRIIIA, without compromising affinity (Routledgeet al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). As a furtherexample of new functionality arising from mutations described aboveaffinity for Fc receptors may be increased beyond that of wild type insome instances. This increased affinity may reflect an increased “on”rate, a decreased “off” rate or both an increased “on” rate and adecreased “off” rate. Examples of mutations believed to impart anincreased affinity for Fc receptors include, but not limited to, T256A,T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

Additionally, at least three human Fc gamma receptors appear torecognize a binding site on IgG within the lower hinge region, generallyamino acids 234-237. Therefore, another example of new functionality andpotential decreased immunogenicity may arise from mutations of thisregion, as for example by replacing amino acids 233-236 of human IgG1“ELLG” to the corresponding sequence from IgG2 “PVA” (with one aminoacid deletion). It has been shown that FcγRI, FcγRII, and FcγRIII, whichmediate various effector functions will not bind to IgG1 when suchmutations have been introduced. Ward and Ghetie 1995, TherapeuticImmunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

In one embodiment, the immunoglobulin constant region or a portionthereof, e.g., an Fc region, is a polypeptide including the sequencePKNSSMISNTP (SEQ ID NO: 98) and optionally further including a sequenceselected from HQSLGTQ (SEQ ID NO: 93), HQNLSDGK (SEQ ID NO: 94),HQNISDGK (SEQ ID NO: 95), or VISSHLGQ (SEQ ID NO: 96) (U.S. Pat. No.5,739,277).

In certain embodiments, the immunoglobulin constant region or a portionthereof is hemi-glycosylated. For example, the fusion protein comprisingtwo Fc regions or FcRn binding partners may contain a first,glycosylated, Fc region (e.g., a glycosylated CH2 region) and a second,aglycosylated, Fc region (e.g., an aglycosylated CH2 region). In oneembodiment, a linker may be interposed between the glycosylated andaglycosylated Fc regions. In another embodiment, the Fc region or FcRnbinding partner is fully glycosylated, i.e., all of the Fc regions areglycosylated. In other embodiments, the Fc region may be aglycosylated,i.e., none of the Fc moieties are glycosylated.

In certain embodiments, a fusion protein of the disclosure comprises anamino acid substitution to an immunoglobulin constant region or aportion thereof (e.g., Fc variants), which alters theantigen-independent effector functions of the Ig constant region, inparticular the circulating half-life of the protein.

Such proteins exhibit either increased or decreased binding to an Fcreceptor when compared to proteins lacking these substitutions and,therefore, have an increased or decreased half-life in serum,respectively. Fc variants with improved affinity for an Fc receptor areanticipated to have longer serum half-lives, and such molecules haveuseful applications in methods of treating mammals where long half-lifeof the administered polypeptide is desired, e.g., to treat a chronicdisease or disorder (see, e.g., U.S. Pat. Nos. 7,348,004, 7,404,956, and7,862,820). In contrast, Fc variants with decreased Fc receptor bindingaffinity are expected to have shorter half-lives, and such molecules arealso useful, for example, for administration to a mammal where ashortened circulation time may be advantageous, e.g., for in vivodiagnostic imaging or in situations where the starting polypeptide hastoxic side effects when present in the circulation for prolongedperiods. Fc variants with decreased Fc receptor binding affinity arealso less likely to cross the placenta and, thus, are also useful in thetreatment of diseases or disorders in pregnant women. In addition, otherapplications in which reduced Fc receptor binding affinity may bedesired include those applications in which localization the brain,kidney, and/or liver is desired. In one exemplary embodiment, the fusionprotein of the disclosure exhibit reduced transport across theepithelium of kidney glomeruli from the vasculature. In anotherembodiment, the fusion protein of the disclosure exhibit reducedtransport across the blood brain barrier (BBB) from the brain, into thevascular space. In one embodiment, a protein with altered Fc receptorbinding comprises at least one Fc region (e.g., one or two Fc regions)having one or more amino acid substitutions within the “Fc receptorbinding loop” of an Ig constant region. The Fc receptor binding loop iscomprised of amino acid residues 280-299 (according to EU numbering) ofa wild-type, full-length, Fc region. In other embodiments, an Igconstant region or a portion thereof of the disclosure having altered Fcreceptor binding affinity comprises at least one Fc region having one ormore amino acid substitutions at an amino acid position corresponding toany one of the following EU positions: 256, 277-281, 283-288, 303-309,313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and438. Exemplary amino acid substitutions which altered Fc receptorbinding activity are disclosed in International PCT Publication No.WO05/047327.

2) Albumin or Fragment, or Variant Thereof

In certain embodiments, the heterologous moiety linked to the IL2polypeptide and/or the IL2Rα EC domain is albumin or a functionalfragment thereof.

Human serum albumin (HSA, or HA), a protein of 609 amino acids in itsfull-length form, is responsible for a significant proportion of theosmotic pressure of serum and also functions as a carrier of endogenousand exogenous ligands. The term “albumin” as used herein includesfull-length albumin or a functional fragment, variant, derivative, oranalog thereof.

In one embodiment, the fusion protein comprises the IL2 polypeptide andthe IL2Rα EC domain described herein and albumin, fragment, or variantthereof, wherein the IL2 polypeptide is linked to albumin or a fragmentor variant thereof. In another embodiment, the fusion protein comprisesthe IL2 polypeptide and the IL2Rα EC domain described herein andalbumin, fragment, or variant thereof, wherein the IL2Rα EC is linked toalbumin or a fragment or variant thereof.

In other embodiments, the heterologous moiety linked to the IL2polypeptide and the IL2Rα EC domain is albumin or a fragment or variantthereof, which extends (or is capable of extending) the half-life of theIL2 polypeptide and the IL2Rα EC domain. Further examples of albumin orthe fragments or variants thereof are disclosed in US Pat. Publ. Nos.2008/0194481 A1, 2008/0004206 A1, 2008/0161243 A1, 2008/0261877 A1, or2008/0153751 A1 or PCT Appl. Publ. Nos. 2008/033413 A2, 2009/058322 A1,or 2007/021494 A2.

3) Albumin Binding Moiety

In certain embodiments, the heterologous moiety linked to the IL2polypeptide and the IL2Rα EC domain is an albumin binding moiety, whichcomprises an albumin binding peptide, a bacterial albumin bindingdomain, an albumin-binding antibody fragment, or any combinationsthereof. For example, the albumin binding protein can be a bacterialalbumin binding protein, an antibody or an antibody fragment includingdomain antibodies (see U.S. Pat. No. 6,696,245). An albumin bindingprotein, for example, can be a bacterial albumin binding domain, such asthe one of streptococcal protein G (Konig, T. and Skerra, A. (1998) J.Immunol. Methods 218, 73-83). Other examples of albumin binding peptidesthat can be used as conjugation partner are, for instance, those havinga Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, H is, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thras described in US patent application 2003/0069395 or Dennis et al.(Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

4) PAS Sequence

In other embodiments, the heterologous moiety linked the IL2 polypeptideand the IL2Rα EC domain is a PAS sequence. In one embodiment, the fusionprotein comprises the IL2 polypeptide and the IL2Rα EC domain describedherein and a PAS sequence, wherein the IL2 polypeptide and/or the IL2RαEC domain is linked to the PAS sequence.

A PAS sequence, as used herein, means an amino acid sequence comprisingmainly alanine and serine residues or comprising mainly alanine, serine,and proline residues, the amino acid sequence forming random coilconformation under physiological conditions. Accordingly, the PASsequence is a building block, an amino acid polymer, or a sequencecassette comprising, consisting essentially of, or consisting ofalanine, serine, and proline which can be used as a part of theheterologous moiety in the fusion protein. Yet, the skilled person isaware that an amino acid polymer also may form random coil conformationwhen residues other than alanine, serine, and proline are added as aminor constituent in the PAS sequence. The term “minor constituent” asused herein means that amino acids other than alanine, serine, andproline may be added in the PAS sequence to a certain degree, e.g., upto about 12%, i.e., about 12 of 100 amino acids of the PAS sequence, upto about 10%, i.e. about 10 of 100 amino acids of the PAS sequence, upto about 9%, i.e., about 9 of 100 amino acids, up to about 8%, i.e.,about 8 of 100 amino acids, about 6%, i.e., about 6 of 100 amino acids,about 5%, i.e., about 5 of 100 amino acids, about 4%, i.e., about 4 of100 amino acids, about 3%, i.e., about 3 of 100 amino acids, about 2%,i.e., about 2 of 100 amino acids, about 1%, i.e., about 1 of 100 of theamino acids. The amino acids different from alanine, serine and prolinemay be selected from the group consisting of Arg, Asn, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val.

Under physiological conditions, the PAS sequence stretch forms a randomcoil conformation and thereby can mediate an increased in vivo and/or invitro stability to the IL2 polypeptide. Since the random coil domaindoes not adopt a stable structure or function by itself, the biologicalactivity mediated by the IL2 polypeptide and the IL2Rα EC domain towhich it is fused is essentially preserved. In other embodiments, thePAS sequences that form random coil domain are biologically inert,especially with respect to proteolysis in blood plasma, immunogenicity,isoelectric point/electrostatic behavior, binding to cell surfacereceptors or internalization, but are still biodegradable, whichprovides clear advantages over synthetic polymers such as PEG.

Non-limiting examples of the PAS sequences forming random coilconformation comprise an amino acid sequence selected from the groupconsisting of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 85), AAPASPAPAAPSAPAPAAPS(SEQ ID NO: 86), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 87),APSSPSPSAPSSPSPASPS (SEQ ID NO: 88), SSPSAPSPSSPASPSPSSPA (SEQ ID NO:89), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 90) and ASAAAPAAASAAASAPSAAA(SEQ ID NO: 91) or any combinations thereof. Additional examples of PASsequences are known from, e.g., US Pat. Publ. No. 2010/0292130 A1 andPCT Appl. Publ. No. WO 2008/155134 A1.

5) HAP Sequence

In certain embodiments, the heterologous moiety linked to the IL2polypeptide and the IL2Rα EC domain is a glycine-rich homo-amino-acidpolymer (HAP). The HAP sequence can comprise a repetitive sequence ofglycine, which has at least 50 amino acids, at least 100 amino acids,120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400amino acids, 450 amino acids, or 500 amino acids in length. In oneembodiment, the HAP sequence is capable of extending half-life of amoiety fused to or linked to the HAP sequence. Non-limiting examples ofthe HAP sequence includes, but are not limited to (Gly)_(n),(Gly₄Ser)_(n) or S(Gly₄Ser)_(n), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, n is20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, or 40. In another embodiment, n is 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, or 200.

6) Transferrin or Fragment Thereof

In certain embodiments, the heterologous moiety linked to the IL2polypeptide and the IL2Rα EC domain is transferrin or a fragmentthereof. Any transferrin may be used to make the fusion proteins of thedisclosure. As an example, wild-type human Tf (Tf) is a 679 amino acidprotein, of approximately 75 KDa (not accounting for glycosylation),with two main domains, N (about 330 amino acids) and C (about 340 aminoacids), which appear to originate from a gene duplication. See GenBankaccession numbers NM001063, XM002793, M12530, XM039845, XM 039847 andS95936 (www.ncbi.nlm.nih.gov/). Transferrin comprises two domains, Ndomain and C domain. N domain comprises two subdomains, N1 domain and N2domain, and C domain comprises two subdomains, C1 domain and C2 domain.

In one embodiment, the transferrin portion of the fusion proteinincludes a transferrin splice variant. In one example, a transferrinsplice variant can be a splice variant of human transferrin, e.g.,Genbank Accession AAA61140. In another embodiment, the transferrinportion of the fusion protein includes one or more domains of thetransferrin sequence, e.g., N domain, C domain, N1 domain, N2 domain, C1domain, C2 domain or any combinations thereof.

7) Polymer, e.g., Polyethylene Glycol (PEG)

In other embodiments, the heterologous moiety attached to the IL2polypeptide and the IL2Rα EC domain is a soluble polymer known in theart, including, but not limited to, polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran, orpolyvinyl alcohol. The heterologous moiety such as soluble polymer canbe attached to any positions within the IL2 polypeptide or the IL2Rα ECdomain or the N- or C-terminus.

Also provided by the disclosure are chemically modified derivatives ofthe fusion protein of the disclosure which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for modification can be selected fromthe group consisting of water soluble polymers including, but notlimited to, polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, and polyvinyl alcohol. Thefusion protein may be modified at random positions within the moleculeor at the N- or C-terminus, or at predetermined positions within themolecule and may include one, two, three or more attached chemicalmoieties.

The polymer can be of any molecular weight, and can be branched orunbranched. For polyethylene glycol, in one embodiment, the molecularweight is between about 1 kDa and about 100 kDa for ease in handling andmanufacturing. Other sizes may be used, depending on the desired profile(e.g., the duration of sustained release desired, the effects, if any onbiological activity, the ease in handling, the degree or lack ofantigenicity and other known effects of the polyethylene glycol to aprotein or analog). For example, the polyethylene glycol may have anaverage molecular weight of about 200, 500, 1000, 1500, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000,13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500,18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000,45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,90,000, 95,000, or 100,000 kDa.

In some embodiments, the polyethylene glycol may have a branchedstructure. Branched polyethylene glycols are described, for example, inU.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol.56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999).

The number of polyethylene glycol moieties attached to each fusionprotein, the IL2 polypeptide, or the IL2Rα EC domain of the disclosure(i.e., the degree of substitution) may also vary. For example, thepegylated proteins of the disclosure may be linked, on average, to 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycolmolecules. Similarly, the average degree of substitution within rangessuch as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13,12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycolmoieties per protein molecule. Methods for determining the degree ofsubstitution are discussed, for example, in Delgado et al., Crit. Rev.Thera. Drug Carrier Sys. 9:249-304 (1992).

In other embodiments, the IL2 polypeptide and the IL2Rα EC domain usedin the disclosure is conjugated to one or more polymers. The polymer canbe water-soluble and covalently or non-covalently attached to the IL2polypeptide, the IL2Rα EC domain or other moieties conjugated to the IL2polypeptide or the IL2Rα EC domain. Non-limiting examples of the polymercan be poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinylalcohol), polyoxazoline, or poly(acryloylmorpholine).

8) Hydroxyethyl Starch (HES)

In certain embodiments, the heterologous moiety linked to the IL2polypeptide and the IL2Rα EC domain is a polymer, e.g., hydroxyethylstarch (HES) or a derivative thereof. In one embodiment, a fusionprotein comprises an IL2 polypeptide described herein and HES, whereinthe IL2 polypeptide and the IL2Rα EC domain is linked to HES.

Hydroxyethyl starch (HES) is a derivative of naturally occurringamylopectin and is degraded by alpha-amylase in the body. HES is asubstituted derivative of the carbohydrate polymer amylopectin, which ispresent in corn starch at a concentration of up to 95% by weight. HESexhibits advantageous biological properties and is used as a bloodvolume replacement agent and in hemodilution therapy in the clinics(Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987); andWeidler et al., Arzneim.-Forschung/Drug Res., 41, 494-498 (1991)).

Amylopectin contains glucose moieties, wherein in the main chainalpha-1,4-glycosidic bonds are present and at the branching sitesalpha-1,6-glycosidic bonds are found. The physical-chemical propertiesof this molecule are mainly determined by the type of glycosidic bonds.Due to the nicked alpha-1,4-glycosidic bond, helical structures withabout six glucose-monomers per turn are produced. The physico-chemicalas well as the biochemical properties of the polymer can be modified viasubstitution. The introduction of a hydroxyethyl group can be achievedvia alkaline hydroxyethylation. By adapting the reaction conditions itis possible to exploit the different reactivity of the respectivehydroxy group in the unsubstituted glucose monomer with respect to ahydroxyethylation. Owing to this fact, the skilled person is able toinfluence the substitution pattern to a limited extent.

HES is mainly characterized by the molecular weight distribution and thedegree of substitution. The degree of substitution, denoted as DS,relates to the molar substitution, is known to the skilled people. SeeSommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), as citedabove, in particular p. 273.

In one embodiment, hydroxyethyl starch has a mean molecular weight(weight mean) of from 1 to 300 kD, from 2 to 200kD, from 3 to 100 kD, orfrom 4 to 70kD. hydroxyethyl starch can further exhibit a molar degreeof substitution of from 0.1 to 3, preferably 0.1 to 2, more preferred,0.1 to 0.9, preferably 0.1 to 0.8, and a ratio between C2:C6substitution in the range of from 2 to 20 with respect to thehydroxyethyl groups. A non-limiting example of HES having a meanmolecular weight of about 130 kD is a HES with a degree of substitutionof 0.2 to 0.8 such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8, preferablyof 0.4 to 0.7 such as 0.4, 0.5, 0.6, or 0.7. In a specific embodiment,HES with a mean molecular weight of about 130 kD is VOLUVEN® fromFresenius. VOLUVEN® is an artificial colloid, employed, e.g., for volumereplacement used in the therapeutic indication for therapy andprophylaxis of hypovolaemia. The characteristics of VOLUVEN® are a meanmolecular weight of 130,000+/−20,000 D, a molar substitution of 0.4 anda C2:C6 ratio of about 9:1. In other embodiments, ranges of the meanmolecular weight of hydroxyethyl starch are, e.g., 4 to 70 kD or 10 to70 kD or 12 to 70 kD or 18 to 70 kD or 50 to 70 kD or 4 to 50 kD or 10to 50 kD or 12 to 50 kD or 18 to 50 kD or 4 to 18 kD or 10 to 18 kD or12 to 18 kD or 4 to 12 kD or 10 to 12 kD or 4 to 10 kD. In still otherembodiments, the mean molecular weight of hydroxyethyl starch employedis in the range of from more than 4 kD and below 70 kD, such as about 10kD, or in the range of from 9 to 10 kD or from 10 to 11 kD or from 9 to11 kD, or about 12 kD, or in the range of from 11 to 12 kD) or from 12to 13 kD or from 11 to 13 kD, or about 18 kD, or in the range of from 17to 18 kD or from 18 to 19 kD or from 17 to 19 kD, or about 30 kD, or inthe range of from 29 to 30, or from 30 to 31 kD, or about 50 kD, or inthe range of from 49 to 50 kD or from 50 to 51 kD or from 49 to 51 kD.

In certain embodiments, the heterologous moiety can be mixtures ofhydroxyethyl starches having different mean molecular weights and/ordifferent degrees of substitution and/or different ratios of C2:C6substitution. Therefore, mixtures of hydroxyethyl starches may beemployed having different mean molecular weights and different degreesof substitution and different ratios of C2:C6 substitution, or havingdifferent mean molecular weights and different degrees of substitutionand the same or about the same ratio of C2:C6 substitution, or havingdifferent mean molecular weights and the same or about the same degreeof substitution and different ratios of C2:C6 substitution, or havingthe same or about the same mean molecular weight and different degreesof substitution and different ratios of C2:C6 substitution, or havingdifferent mean molecular weights and the same or about the same degreeof substitution and the same or about the same ratio of C2:C6substitution, or having the same or about the same mean molecularweights and different degrees of substitution and the same or about thesame ratio of C2:C6 substitution, or having the same or about the samemean molecular weight and the same or about the same degree ofsubstitution and different ratios of C2:C6 substitution, or having aboutthe same mean molecular weight and about the same degree of substitutionand about the same ratio of C2:C6 substitution.

9) Polysialic Acids (PSA)

In certain embodiments, the non-polypeptide heterologous moiety linkedto the IL2 polypeptide and/or the IL2Rα EC domain is a polymer, e.g.,polysialic acids (PSAs) or a derivative thereof. Polysialic acids (PSAs)are naturally occurring unbranched polymers of sialic acid produced bycertain bacterial strains and in mammals in certain cells Roth J., etal. (1993) in Polysialic Acid: From Microbes to Man, eds Roth J.,Rutishauser U., Troy F. A. (Birkhauser Verlag, Basel, Switzerland), pp335-348. They can be produced in various degrees of polymerisation fromn=about 80 or more sialic acid residues down to n=2 by limited acidhydrolysis or by digestion with neuraminidases, or by fractionation ofthe natural, bacterially derived forms of the polymer. The compositionof different polysialic acids also varies such that there arehomopolymeric forms i.e. the alpha-2,8-linked polysialic acid comprisingthe capsular polysaccharide of E. coli strain K1 and the group-Bmeningococci, which is also found on the embryonic form of the neuronalcell adhesion molecule (N-CAM). Heteropolymeric forms also exist—such asthe alternating alpha-2,8 alpha-2,9 polysialic acid of E. coli strainK92 and group C polysaccharides of N. meningitidis. Sialic acid may alsobe found in alternating copolymers with monomers other than sialic acidsuch as group W135 or group Y of N. meningitidis. Polysialic acids haveimportant biological functions including the evasion of the immune andcomplement systems by pathogenic bacteria and the regulation of glialadhesiveness of immature neurons during foetal development (wherein thepolymer has an anti-adhesive function) Cho and Troy, P.N.A.S., USA, 91(1994) 11427-11431, although there are no known receptors for polysialicacids in mammals. The alpha-2,8-linked polysialic acid of E. coli strainK1 is also known as ‘colominic acid’ and is used (in various lengths) toexemplify the present disclosure. Various methods of attaching orconjugating polysialic acids to a polypeptide have been described (forexample, see U.S. Pat. No. 5,846,951; WO-A-0187922, and US 2007/0191597A1.

10) XTEN Sequences

As used here “XTEN sequence” refers to extended length polypeptides withnon-naturally occurring, substantially non-repetitive sequences that arecomposed mainly of small hydrophilic amino acids, with the sequencehaving a low degree or no secondary or tertiary structure underphysiologic conditions. As a fusion protein partner, XTENs can serve asa carrier, conferring certain desirable pharmacokinetic, physicochemicaland pharmaceutical properties when linked to the IL2 polypeptide and/orthe IL2Rα EC domain of the disclosure to create a fusion protein. Suchdesirable properties include but are not limited to enhancedpharmacokinetic parameters and solubility characteristics.

In some embodiments, the XTEN sequence of the disclosure is a peptide ora polypeptide having greater than about 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acidresidues. In certain embodiments, XTEN is a peptide or a polypeptidehaving greater than about 20 to about 3000 amino acid residues, greaterthan 30 to about 2500 residues, greater than 40 to about 2000 residues,greater than 50 to about 1500 residues, greater than 60 to about 1000residues, greater than 70 to about 900 residues, greater than 80 toabout 800 residues, greater than 90 to about 700 residues, greater than100 to about 600 residues, greater than 110 to about 500 residues, orgreater than 120 to about 400 residues.

The XTEN sequence of the disclosure can comprise one or more sequencemotif of 9 to 14 amino acid residues or an amino acid sequence at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe sequence motif, wherein the motif comprises, consists essentiallyof, or consists of 4 to 6 types of amino acids selected from the groupconsisting of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E) and proline (P). See US 2010-0239554 A1.

In some embodiments, an XTEN sequence comprises multiple units ofnon-overlapping sequence motifs of the AD motif family, or of the AEmotif family, or of the AF motif family, or of the AG motif family, orof the AM motif family, or of the AQ motif family, or of the BC family,or of the BD family, with the resulting XTEN exhibiting the range ofhomology. In other embodiments, the XTEN comprises multiple units ofmotif sequences from two or more of the motif families. These sequencescan be selected to achieve desired physical/chemical characteristics,including such properties as net charge, hydrophilicity, lack ofsecondary structure, or lack of repetitiveness that are conferred by theamino acid composition of the motifs, described more fully below. Inother embodiments, the motifs incorporated into the XTEN can be selectedand assembled using the methods described herein to achieve an XTEN ofabout 36 to about 3000 amino acid residues.

In further embodiments, the XTEN sequence used in the disclosure affectsthe physical or chemical property, e.g., pharmacokinetics, of the fusionprotein of the present disclosure. The XTEN sequence used in the presentdisclosure can exhibit one or more of the following advantageousproperties: conformational flexibility, enhanced aqueous solubility,high degree of protease resistance, low immunogenicity, low binding tomammalian receptors, or increased hydrodynamic (or Stokes) radii. In aspecific embodiment, the XTEN sequence linked to the IL2 polypeptideand/or the IL2Rα EC domain in this disclosure increases pharmacokineticproperties such as longer terminal half-life or increased area under thecurve (AUC), so that the fusion protein described herein stays in vivofor an increased period of time compared to wild type the IL2polypeptide. In further embodiments, the XTEN sequence used in thisdisclosure increases pharmacokinetic properties such as longer terminalhalf-life or increased area under the curve (AUC), so that the IL2polypeptide stays in vivo for an increased period of time compared towild type IL2.

A variety of methods and assays can be employed to determine thephysical/chemical properties of proteins comprising the XTEN sequence.Such methods include, but are not limited to analytical centrifugation,EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, lightscattering, capillary electrophoresis, circular dichroism, differentialscanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-sizeexclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visiblespectroscopy. Additional methods are disclosed in Amau et al., Prot Exprand Purif 48, 1-13 (2006).

Additional examples of XTEN sequences that can be used according to thepresent disclosure and are disclosed in US Patent Publication Nos.2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1,2011/0077199 A1, or 2011/0172146 A1, or International Patent PublicationNos. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228A1, WO 2011028229 A1, or WO 2011028344 A2.

11) Immunoglobulin Binding Peptide (or Polypeptide)

In certain embodiments, the non-heterologous moiety linked to the IL2polypeptide and/or the IL2Rα EC domain is an immunoglobulin bindingpeptide. The immunoglobulin binding peptides can bind to an Fc regionand can improve a half-life of the fusion protein described herein.

In some embodiments, the immunoglobulin binding peptide useful for thedisclosure is a peptide or a polypeptide having greater than about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acidresidues.

In some embodiments, the immunoglobulin binding peptide useful for thedisclosure comprises a 13-mer IgG-Fc domain binding peptide (IgGBP).DeLano W L, et al. (2000) Science 287:1279-1283. In other embodiments,the immunoglobulin binding peptide useful for the disclosure comprisesthe peptides disclosed in US Patent Publication No. 20170334954, USPatent Publication No. 20170210777, or PCT Publication No.WO/2017/069158.

7.3.5 Fusion Protein

In some embodiments, the fusion proteins comprise any one of SEQ IDNO:13 to SEQ ID NO:70 and SEQ ID NO:202 to SEQ ID NO:204 as recited inTable 3.

TABLE 3 SEQ ID NO: Construct Sequence 204 IL2(21-153)-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH (G3S)3-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE CD25(22-240)TATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQ  13 IL2(C145S)-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-240)LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQ  14 IL2(C145A)-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-240)LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQ  15 IL2(C145V)-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-240)LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFVQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQ  16 IL2-CD25(22-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH 212)LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS  17IL2-CD25(22- APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH187) LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  18 IL2(C145A)-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(C213S,LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE 22-240)TATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAATMETSIFTTEYQ  19 HSA-(G₄S)₃-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVA IL2-CD25(22-DESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNP 187)NLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG E  20IL2(C145A)- APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHCD25(22-212)- LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADEGG-HSA TATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSGGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA ASQAALGL  21IL2(C145A)- APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHCD25(22-192)- LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADEGGC TATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQGGC  22 IL2(C145A)-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-213)LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETSC  23IL2(C145A)- APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHCD25(22-187, LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADEN70C) TATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGCSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  24 IL2(C145A)-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-187,LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE N89C)TATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRCTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  25 IL2(C145A)-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-187)-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE CTATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEC  26 Human DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Fc1.1(f)-AZ1-NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPS IL2(C145S)-SIEKTISKAKGQPREPQVYVYPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG CD25(22-187)-QPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS GLSLSPGAAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPK first side of KATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTF Fc to make asMCEYADETATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHA heterodimericTFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEG  27 IL2(C145S)-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-187)-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE HuFc1.1(f)-AZ1TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAY first side of KEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP Fc to make asQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQ heterodimericCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEPKSSDKTHTCPPCPAPEAE pairGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYVYPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG  29 IL2-C145S-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-187)LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  30 IL2-T23A-APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH C145S-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE CD25(22-187)TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  31 IL2-CD25(22-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH 240)-C213SLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAATMETSIFTTEYQ  32 IL2-T23A-APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH C145S-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE CD25(22-240)-TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAY C213SKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAATMETSIFTTEYQ  33 Native SigPep-MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK IL2-CD25(22-LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN 187)VIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG E  34Native SigPep- MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKHuIL2- LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINCD25(22-212)- VIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGS PPELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSPP  35 M-IL2(C145S)-MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK CD25(C213S)HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAATMETSIFTTEYQ  36 M-IL2(C145S)-MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK CD25(C213S)HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAATMETSIFTTEYQ  37 M-IL2-C145A-MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK CD25(22-212)HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEG RPESETS  38M-IL2-C145A- MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKCD25(22-212) HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEG RPESETS  39Del-A21-IL2- PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKHLCD(22-212) QCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRP ESETS  40IL2-T23A-L2- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKHCD25(22-212) LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGSSGGAGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS  41IL2-T3A-L3- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKHCD(22-212) LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEK PQASPEGRPESETS 43 IL2-T23A- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKHC145S- LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADECD25(22-187) TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  44 IL2-T23A-APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKH C145S-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE CD25(22-240)-TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAY C213SKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAATMETSIFTTEYQ  45 IL2-T23A-APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKH CD25(22-187)LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  46 IL2-T23A-APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKH CD25(22-212)-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE T106ATATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKATEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS  47IL2-T23A- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKHCD25(22-212)- LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET95A TATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVAPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS  49IL2-T23A- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKHCD25(22-212) LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS  50IL2-T23A- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKH C145S-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE CD25(22-212)TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS  51IL2-C145S- APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKHCD25(22-212) LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS  52IL2-T23A- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKHCD25(22-212, LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET95A-T105A) TATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVAPQPEEQKERKATEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS  53IL2-T23A- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKHCD25(22-212)- LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE PGTATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETSPG  54IL2-CD25(22- APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKH187) LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  55 IL2-T23A-APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKEYMPKKATELKH C145S-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE CD25(22-187)TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  56 IL2-CD25(22-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH 240, C213S)LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAATMETSIFTTEYQ  57 Del-A21-IL2-PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHL CD(22-212)QCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRP ESETS  58IL2-T23A-L2- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHCD25(22-212) LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGSSGGAGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS  59IL2-T23A-L3- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHCD25(22-212) LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEK PQASPEGRPESETS 60 IL2-T23A- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHC145S- LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADECD25(22-187)- TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYN70Q-N89Q KEGTMLNCECKRGFRRIKSGSLYMLCTGQSSHSSWDNQCQCTSSATRQTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  61 IL2-C145S-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-187)-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE N70Q-N89QTATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGQSSHSSWDNQCQCTSSATRQTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  62 IL2-T23A-APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH C145S-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE CD25(22-187)-TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAY N89QKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRQTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  63 IL2-T23A -APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH C145S-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE CD25(22-187)-TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAY N70QKEGTMLNCECKRGFRRIKSGSLYMLCTGQSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  64 IL2-C145S-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-187)-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE N89QTATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRQTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  65 IL2-C145S-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH CD25(22-187)-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE N70QTATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGQSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  66 IL2-T23A -APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH C145S-LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE CD25(22-187)TATIVEFLNRWITFSQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE  67 Fc1.1-7linker-DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF IL2-CD25(22-NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPS 212)SIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG Generates FcQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS homodimerLSLSPGGSGGSGGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETS  68 IL2-CD25(22-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH 212)-Fc1.1LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE Generates FcTATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAY homodimerKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG  69IL2(V111K)- APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHCD25(22-212) LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINKIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS  70IL2(D40T)- APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHCD25(22-212) LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGR PESETS 202IL2-T3A- APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHCD25(22-187)- LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADEN89Q-T95A- TATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAY T106AKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRQTTKQVAPQPEEQKERKATEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE 203 Fc-DKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF (IL2(V91K)CD2NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA 5)2 bivalentPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSGGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINKIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETS

IL2 is mature (IL2(21-153) unless noted otherwise. Linker between IL2and CD25 is (G3S)3 unless otherwise noted.

In some embodiments, the fusion protein of the present disclosurecomprises an amino acid sequence at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, or about 100%identical to any one of SEQ ID NO:13 to SEQ ID NO:70 and SEQ ID NO:202to SEQ ID NO:204.

The IL2-IL2Rα fusion protein of the present disclosure can have one ormore the following properties/activities: (1) increasing activity ofregulatory T cells (Tregs) and/or increasing immune tolerance in lowdose IL2 based therapies; (2) increasing immune response and memory inhigher dose therapies; (3) increasing IL2 availability when compared torecombinant IL2; and/or (4) increasing persistent IL2 stimulation ofIL2R bearing lymphocytes in vivo.

In one embodiment, the fusion proteins disclosed herein comprises one ormore pharmacokinetic properties selected from the group consisting of anincreased half-life, increased C_(max), increased AUC, increasedC_(min), decreased clearance, improved bioavailability, and anycombination thereof, compared to the pharmacokinetic property of thepolypeptide consisting of IL2 (SEQ ID NO:2) or SEQ ID NO:204 (wtIL2-CD25 sequence with the 12mer linker without truncation).

In one embodiment, the fusion proteins disclosed herein have an extendedhalf-life compared to IL2 (SEQ ID NO:2) or SEQ ID NO:204 (wt IL2-CD25sequence with the 12mer linker without truncation). In some embodiments,the extended half-life is at least about 1.5 fold, at least about 2fold, at least about 3 fold, at least about 4 fold, at least about 5fold, at least about 6 fold, at least about 7 fold, at least about 8fold, at least about 9 fold, at least about 10 fold, at least about 11fold, at least about 12 fold, at least about 13 fold, at least about 14fold, at least about 15 fold, at least about 16 fold, at least about 17fold, at least about 18 fold, at least about 19 fold, at least about 20fold, at least about 21 fold, or at least about 22 fold compared to thehalf-life of a polypeptide consisting of IL2 (SEQ ID NO:2) or SEQ ID NO:204 (wt IL2-CD25 sequence with the 12mer linker without any truncation).

In some embodiments, an increased activity of Tregs that results fromthe IL2/IL2Rα fusion protein can be assayed in a variety of waysincluding, for example, (1) an increased representation and number ofTregs in the CD4+ T cell compartment; (2) upregulation of IL2-dependentCD25; (3) increased proliferation as assessed by expression of theproliferative marker Ki67; and (4) an increased fraction ofIL2-dependent terminally differentiated Klrg1+ Treg subset. Such effectson Tregs can be seen in, for example, in the spleen and/or the inflamedpancreas.

In some embodiments, the IL2/IL2Rα fusion protein of the presentdisclosure increases tolerogenic and immune suppressive Tregs andimmunity through increasing T effector/memory responses and, in furtherembodiments, it exhibits improved pharmacokinetics by delivering suchresponses at (1) lower effective levels of IL2 activity compared tonative or recombinant IL2; and/or (2) displays more persistentbiological responses than native or recombinant IL2.

In specific embodiments, the fusion protein has an improved activityover the native or recombinant IL2. For example, the effect of theIL2/IL-2Rα fusion protein can increase tolerogenic Tregs at about 2fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70fold, 80 fold, 90 fold, 100 fold 150 fold, 200 fold or lower level IL2activity in comparison to native or recombinant IL2. In otherembodiments, the IL2/IL2Rα fusion protein is more effective than nativeor recombinant IL2 in inducing persistent augmentation of Tregs andrelated properties.

Various IL2 and IL2Rα fragments and variants from a variety of organismcan be used to generate the IL2/IL2Rα extracellular domain fusionproteins provided herein. Such components are discussed in furtherdetailed elsewhere herein. Examples of non-limiting unprocessed ormature IL2/IL2Rα extracellular domain fusion proteins are set forth inSEQ ID NOs: SEQ ID NO:13 to SEQ ID NO:70 and SEQ ID NO:202 to SEQ IDNO:204.

The term “secretory signal sequence” denotes a polynucleotide sequencethat encodes a polypeptide (a “secretory peptide”) that, as a componentof a larger polypeptide, directs the larger polypeptide through asecretory pathway of the cell in which it is synthesized. The largerpolypeptide is commonly cleaved to remove the secretory peptide duringthe transit through the secretory pathway. As used herein, a “mature”form of a fusion protein or polypeptide comprises the processed form ofthe polypeptide that has had the secretory peptide removed. As usedherein, the “unprocessed” form of the fusion protein retains thesecretory peptide sequence.

Biologically active fragments and variants of the mature and unprocessedform of the IL2/IL-Ra EC domain fusion proteins, and the polynucleotideencoding the same, are also provided. Such a functional polypeptidefragment can comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500 or more continuous amino acids of any one of SEQ ID NOs: SEQ IDNO:13 to SEQ ID NO:70 and SEQ ID NO:202 to SEQ ID NO:204. Alternatively,a functional polypeptide variant can comprise at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity tothe sequence set forth in SEQ ID NOs: SEQ ID NO:13 to SEQ ID NO:70 andSEQ ID NO:202 to SEQ ID NO:204.

Active variants and fragments of polynucleotides encoding the IL2/IL-Raextracellular domain fusion proteins are further provided. Suchpolynucleotide can comprise at least 100, 200, 300, 400, 500, 600, 700,800, 1000, 1100, 1200, 1300, 1500, 1800,2000 continuous nucleotidesencoding the polypeptides set forth in SEQ ID NOs: SEQ ID NO:13 to SEQID NO:70 and SEQ ID NO:202 to SEQ ID NO:204 and continue to encode afunctional IL2/IL-Ra extracellular domain fusion protein.

It is further recognized that the components of the IL2/IL2Rα fusionprotein can be found any order. In one embodiment, the IL2 polypeptideis at the N-terminus and the extracellular domain of IL2Rα is at theC-terminus of the fusion protein.

In some embodiments, the fusion protein forms a dimer. In otherembodiments, the fusion protein is a monomer. Still, in someembodiments, the dimer comprises two monomers, and the monomers areassociated with each other via covalent bonds. In some embodiments, thedimer comprises two monomers, and the monomers are associated vianon-covalent bonds.

In some embodiment of the disclosure, the fusion protein is more stablethan the polypeptide consisting of IL2 (SEQ ID NO: 2) or SEQ ID NO: 204(wt IL2-CD25 sequence with the 12mer linker without truncation). In someembodiments, the fusion protein has one or more properties selected fromthe group consisting of (i) increased thermodynamic stability comparedto a reference protein; (ii) increased TM compared to a referenceprotein; (iii) increased resistant to degradation compared to areference protein; (iv) increased resistance to modifications comparedto a reference protein; (v) increased stability in vivo compared to areference protein; and (vi) any combination thereof, wherein thereference protein comprises (i) a first polypeptide comprising anInterleukin-2 (IL2) polypeptide; and (b) a second polypeptide comprisingan extracellular domain of an Interleukin-2 Receptor alpha (IL2Rα)polypeptide; and has at least one more glycosylation compared to thefusion protein.

Any of the glycosylation sites of the fusion proteins disclosed hereincan be removed by other mechanisms. In some embodiments, the fusionprotein is deglycosylated enzymatically or chemically. In someembodiments, the fusion protein is deglycosylated by alkali,hydrazinolysis, Peptide-N-Glycosidase F (PNGase F),Endo-β-N-acetylglucosaminidase H (Endo H), O-glycosidase, or anycombination thereof.

In some embodiments, removal of one or more glycosylation sites of thefusion protein is achieved by treatment of the fusion protein with analkali. In some embodiments, the glycans are removed from theglycosylated polypeptides by alkali borohydride treatment. In otherembodiments, glycosylation sites of the fusion proteins disclosed hereincan be removed using alkaline metal carbonates such as sodium carbonateand potassium carbonate. In some embodiments, the alkali is used forβ-elimination treatment.

In some embodiments, removal of one or more glycosylation sites of thefusion protein is achieved by chemical treatment of the fusion proteinby means of hydrazinolysis. In one embodiment, glycosylations arereleased from a fusion protein disclosed herein by subjecting the fusionprotein to hydrazinolysis, and the released sugar chain is subjected tofluorescence labeling with 2-aminopyridine. See Hase et al. J. Biochem.,95, 197 (1984). In some embodiments, hydrazinolysis is carried out usingan instrument supplied by Oxford GlycoSystems (the GlycoPrep 1000).

In another embodiment, removal of one or more glycosylation sites of thefusion protein is achieved by subjecting the fusion protein totrifluoromethanesulfonic acid (TFMS).

In some embodiments, removal of one or more glycosylation sites of thefusion protein is achieved by treatment of the fusion protein with anenzyme. In some embodiments, the enzyme is a glycosidase. In someembodiments, removal of one or more glycosylation sites of the fusionprotein is achieved using Peptide-N-Glycosidase F (PNGase F). Theconcentration of PNGase F can vary and is to be determined empirically.In some embodiments, the glycosidase is PNGase F. PNGase F is acommercially available enzyme (e.g., New England Biolabs, Ipswich Mass.,Cat. #P0704 or #P0710). In some embodiments, the PNGase F is a fusionprotein. For example, the PNGase F can be PNGase F tagged with a chitinbinding domain (CBD) or a PNGase F-SNAP fusion protein. In someembodiments, the glycosidase is lyophilized. In some embodiments, theglycosidase is a lyophilized PNGase F. In some embodiments, theglycosidase is substantially free of animal-derived reagents.

In some embodiments, removal of one or more glycosylation sites of thefusion protein is achieved by treatment of the fusion protein withEndo-β-N-acetylglucosaminidase H (Endo H). Endo-H is a glycohydrolasethat is secreted by Streptomyces plicatus and a few other Streptomycesspecies (Tarentino et al., 1976). It cleaves the β-1, 4-glycosidic bondof the N-acetyl glucosamine core of oligosaccharides and leaves oneN-acetylchitobiose attached to the asparagine residue of theglycoprotein (Trimble et al., 1978; Muramatsu 1971). The Endo H gene ofS. plicatus is 939 bp (GenBank accession AAA26738.1) encodes a 28.9-kDaprotein. Endo H from S. plicatus was recently expressed in Pichiapastoris and deglycosylated activity of P. pastoris produced Endo H wasdemonstrated in vitro, through both co-fermentation andpost-fermentation treatments (Wang et al., 2015).

In some embodiments, removal of one or more glycosylation sites of thefusion protein is achieved by treatment of the fusion protein withO-glycosidase (New England Biolabs, Ipswich Mass.). O-glycosides, alsocalled endo-alpha-N-acetylgalactosaminidase, catalyzes the removal ofCore 1 and Core 3 O-linked disaccharides from glycoproteins. In someembodiments, it releases unsubstituted Ser- and Thr-linked fromglycoproteins.

The removal of one or more glycosylation sites of the fusion protein canbe achieved after the IL2/IL2Rα fusion protein is produced in a cellculture (e.g., bioreactor), while the IL2/IL2Rα fusion protein isproduced in a cell culture, after the fusion protein is harvested,and/or while the fusion protein is being purified. In some embodiments,the removal of one or more glycosylation sites can be achieved by addingone or more removal agents during the cell culture while the fusionprotein is expressed. In other embodiments, the removal of one or moreglycosylation sites can be achieved by selecting a particular cell typeas a host cell that eliminates glycosylation or has reducedglycosylation (e.g., E. coli or Streptomyces species). In certainembodiments, the removal of one or more glycosylation sites is achievedby co-expressing a gene encoding the fusion protein with a gene encodingan enzyme that removes one or more glycosylation.

Table 4 below recites various IL-2 amino acid sequences. In someembodiments, the fusion proteins described herein comprises any one ofSEQ ID NO:101 to SEQ ID NO:115 as recited in Table 4.

TABLE 4 SEQ ID Con- NO: struct Sequence 101 IL2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK (21-153)LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 102 IL2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK(21-153) LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL (C145S)NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFSQSIISTLT 103 IL2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK (21-153)LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL (C145A)NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 104 IL2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK (21-153)LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL (C145V)NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFVQSIISTLT 105 IL2APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPK (21-153)LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL (T23A)NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITESQSIISTLT 106 IL2APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPK (21-153)LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL (T23A +NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC C145S) EYADETATIVEFLNRWITFSQSIISTLT107 IL2 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPK (21-153)LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL (T23A +NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC C145A) EYADETATIVEFLNRWITFAQSIISTLT108 Native MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLE SigPep-HLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA IL2TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDL (1-153)ISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT 109 M-IL2MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP (C145S)KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEV LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 110 M-IL2MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP (C145A)KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEV LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 111 Del-PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKL A21-IL2TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 112 Del- PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLA21-IL2 TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN (C145S)LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFSQSIISTLT 113 Del-PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKL A21-IL2TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN (C145A)LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFAQSIISTLT 114 IL2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK (V111K)LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVL NLAQSKNFHLRPRDLISNINKIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 115 IL2 APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPK(D40T) LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFCQSIISTLT

Table 5 below recites various linker amino acid sequences. In someembodiments, a fusion protein disclosed herein includes multipleconcatenated sequences selected from any one of SEQ ID NO:116 to SEQ IDNO:127 as recited in Table 5.

TABLE 5 SEQ ID NO: Construct Sequence 116 L1-(G3S)3 GGGSGGGSGGGS 117L2-G2S2 GGSSGGAGGGGS 118 L3-(G3S)5 GGGSGGGSGGGSGGGSGGGS 119 L4-(G2S)3GGSGGSGGS 120 L5-(G2S)5 GGSGGSGGSGGSGGS 121 L6-GGEEE GGEEEGGEEEGS 122L7-12mer_Stiff ESPEPETPEDES 123 L8- GRGGEEKKKEKEKEEQEERETK 22mer_Helix124 L9-G G 125 L10-GG GG 126 L11-S s 127 L12-GS GS

Table 6 below recites various CD25 amino acid sequences. In someembodiments, the fusion proteins described herein comprises any one ofSEQ ID NO:128 to SEQ ID NO:169 as recited in Table 6.

TABLE 6 SEQ ID NO: Construct Sequence 128 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 240)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQ 129 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 212)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETS 130 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 187)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE 131 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 240)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE (C213S)RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAATMETSIFTTEYQ 132 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 192)-GGCCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQGGC 133 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 213)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSC 134 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGCSSHSSWDNQ 187)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE (N70C)RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE 135 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 187)CQCTSSATRCTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE (N89C)RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE 136 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 187)-CCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEC 137 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 187)-GCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEG 138 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 212)-PPCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSPP 139 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 212)CQCTSSATRNTTKQVTPQPEEQKERKATEMQSPMQPVDQASLPGHCREPPPWENEATE (T106A)RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETS 140 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 212)CQCTSSATRNTTKQVAPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE (T95A)RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETS 141 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 212)CQCTSSATRNTTKQVAPQPEEQKERKATEMQSPMQPVDQASLPGHCREPPPWENEATE (T95A +RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG T106A)EEKPQASPEGRPESETS 142 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 212)-PGCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSPG 143 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGQSSHSSWDNQ 187)CQCTSSATRQTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE (N70Q +RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE N89Q) 144 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 187)CQCTSSATRQTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE (N89Q)RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE 145 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGQSSHSSWDNQ 187)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE (N70Q)RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE 146 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 211)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESET 147 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 210)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESE 148 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 209)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPES 149 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 208)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQASPEGRPE150 CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ207) CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQASPEGRP151 CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ206) CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQASPEGR152 CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ205) CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQASPEG153 CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ204) CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQASPE 154CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 203)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQASP 155CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 202)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQAS 156CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 201)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQA 157CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 200)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQ 158CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 199)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKP 159CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 198)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEK 160CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 197)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EE 161CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 196)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG E 162CD25(22- ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 195)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG 163 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 194)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFP 164 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 193)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQF 165 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 192)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQ 166 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 191)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETS 167 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 190)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMET 168 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 189)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEME 169 CD25(22-ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ 188)CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEM

Table 7 below recites various linker amino acid sequences. In someembodiments, the fusion proteins described herein comprises any one ofSEQ ID NO:170 to SEQ ID NO:186 as recited in Table 7. In someembodiments, there is no linker sequences (i.e., “TL0” as recited inTable 7).

TABLE 7 SEQ ID NO: Construct Sequence TL0 — 170 TL1 G 171 TL2 GG 172 TL3GS 173 TL4 GGS 174 TL5 GGGS 175 TL6 GGSGG 176 TL7 GGGSGG 177 TL8 GGSGGSG178 TL9 GGSGGGSG 179 TL10 GGGSGGGSG 180 TL11 GGGGSGGGGS 181 TL12GGGGSGGGGSG 182 TL13 EPKSS 183 TL14 PKSS 184 TL15 KSS 185 TL16 SS 186TL17 S

Table 8 below recites various enhancer amino acid sequences. In someembodiments, the fusion proteins described herein comprises any one ofSEQ ID NO:187 to SEQ ID NO:201 as recited in Table 8.

TABLE 8 SEQ ID NO: Construct Sequence 187 IgGI-FcDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 188 IgG1.1-FcDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 189 IgG1.3-FcDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 190 IgG1-FcDKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMI (P238K)SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 191IgG4-Fc ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 192IgG4.1-Fc ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 193IgG1.3-Fc- DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMI knobSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 194IgG1.3-Fc- DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMI holeSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 195IgG1-Fc DKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMI (P238K)-SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN knob AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 196IgG1-Fc DKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMI (P238K)-SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN hole AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 197IgG1.1-Fc DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMI (AZ1)SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYVYP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 198IgG1.1-Fc DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMI (AZ2)SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYVLP PSREEMTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 199 HSADAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQ CPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERN ECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAF TECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEV SKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDE MPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAA ADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRN LGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGKKLVAASQAALGL 200 HSA(C34S)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQ SPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERN ECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAF TECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEV SKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDE MPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAA ADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRN LGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGKKLVAASQAALGL 201 HSA(C35A)DAHKSEVAHREKDLGEENFKALVLIAFAQYLQQ APFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERN ECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAF TECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEV SKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDE MPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAA ADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRN LGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGKKLVAASQAALGL

In some embodiments, a fusion protein disclosed herein comprises an IL-2sequence selected from Table 4 (i.e., one of SEQ ID NO:101 to SEQ IDNO:115) and a CD25 sequence from Table 6 (i.e., one of SEQ ID NO:128 toSEQ ID NO:169). In some embodiments, a fusion protein also comprises asequence, or multiple concatenated sequences, chosen from Table 5 (i.e.,one or more of SEQ ID NO:116 to SEQ ID NO:127).

In some embodiments, a fusion protein disclosed herein comprises an IL-2sequence selected from Table 4 (i.e., one of SEQ ID NO:101 to SEQ IDNO:115) above and a CD25 sequence from Table 6 (i.e., one of SEQ IDNO:128 to SEQ ID NO:169), a sequence, or multiple concatenatedsequences, chosen from Table 5 (i.e., one or more of SEQ ID NO:116 toSEQ ID NO:127), and an optional linker comprising a sequence, ormultiple concatenated sequences, from Table 7 (i.e., one or more of SEQID NO:170 to SEQ ID NO:186).

In some embodiments, a fusion protein disclosed herein comprises an IL-2sequence selected from Table 4 (i.e., one of SEQ ID NO:101 to SEQ IDNO:115) above and a CD25 sequence from Table 6 (i.e., one of SEQ IDNO:128 to SEQ ID NO:169), a sequence, or multiple concatenatedsequences, chosen from Table 5, and an optional linker comprising asequence, or multiple concatenated sequences, from Table 8 (i.e., one ofSEQ ID NO:187 to SEQ ID NO:201).

In some embodiments, a fusion protein disclosed herein comprises, inorder, an enhancer sequence from Table 8 (i.e., one of SEQ ID NO:187 toSEQ ID NO:201), a sequence, or multiple concatenated sequences, chosenfrom Table 7 (i.e., one or more of SEQ ID NO:170 to SEQ ID NO:186), anIL-2 sequence selected from Table 4 (i.e., one of SEQ ID NO:101 to SEQID NO:115), and a CD25 sequence from Table 6 (i.e., one of SEQ ID NO:128to SEQ ID NO:169). In some embodiments, the fusion protein comprises asequence, or multiple concatenated sequences, chosen from Table 7 (i.e.,one or more of SEQ ID NO:170 to SEQ ID NO:186). In some embodiments, thefusion protein comprises a sequence, or multiple concatenated sequences,chosen from Table 5 (i.e., one or more of SEQ ID NO:116 to SEQ IDNO:127).

7.4 Polynucleotides

In certain aspects, provided herein are polynucleotides, e.g., DNA orRNA, comprising a nucleotide sequence encoding a fusion proteindescribed herein that has IL2 activity, as well as vectors comprisingsuch polynucleotide sequences, e.g., expression vectors for theirefficient expression in host cells, e.g., mammalian cells. In someembodiments, provided herein are polynucleotide sequences that encodepolypeptide sequences of SEQ ID NO: 1 to SEQ ID NO:70 and SEQ ID NO: 202to SEQ ID NO: 204.

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular lessthan about 10%) of other material, e.g., cellular material, culturemedium, other nucleic acid molecules, chemical precursors and/or otherchemicals. In a specific embodiment, a nucleic acid molecule(s) encodinga fusion protein described herein is isolated or purified.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding fusion proteins described herein, e.g., the fusionproteins described in Table 3, and modified versions of these fusionproteins can be determined using methods well known in the art, i.e.,nucleotide codons known to encode particular amino acids are assembledin such a way to generate a nucleic acid that encodes the fusionprotein. Such a polynucleotide encoding the fusion protein can beassembled from chemically synthesized oligonucleotides (e.g., asdescribed in Kutmeier G et al., (1994), BioTechniques 17: 242-6), which,briefly, involves the synthesis of overlapping oligonucleotidescontaining portions of the sequence encoding the fusion protein,annealing and ligating of those oligonucleotides, and then amplificationof the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding a fusion protein describedherein can be generated from nucleic acid from a suitable source (e.g.,a hybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the fusion protein of interest. Such PCR amplification methodscan be used to obtain nucleic acids comprising the sequence encodinge.g., IL2, a linker sequence, or IL2-Rα. The amplified nucleic acids canbe cloned into vectors for expression in host cells and for furthercloning, for example, to generate fusion proteins.

If a clone containing a nucleic acid encoding a particular fusionprotein is not available, but the sequence of the fusion proteinmolecule is known, a nucleic acid encoding the fusion protein can bechemically synthesized or obtained from a suitable source (e.g., a cDNAlibrary or a cDNA library generated from, or nucleic acid, preferablypoly A+ RNA, isolated from, any tissue or cells expressing the proteinsof interest, such as hybridoma cells selected to express a fusionprotein described herein) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes the fusionproteins. Amplified nucleic acids generated by PCR can then be clonedinto replicable cloning vectors using any method well known in the art.

DNA encoding fusion proteins described herein can be readily isolatedand sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the fusion proteins disclosed herein). Hybridoma cells canserve as a source of such DNA. Once isolated, the DNA can be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells(e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells thatdo not otherwise produce immunoglobulin protein, to obtain the synthesisof fusion proteins in the recombinant host cells.

It is further recognized that the polynucleotide encoding the IL2/IL2Rαfusion protein can comprise additional elements that aid in thetranslation of the fusion protein. Such sequences include, for example,Kozak sequences attached to the 5′ end of the polynucleotide encodingthe fusion protein. The Kozak consensus sequence is a sequence whichoccurs on eukaryotic mRNA that plays a role in the initiation of thetranslation process and has the consensus (gee)gccRccAUGG (SEQ IDNO:92); wherein (1) a lower case letter denotes the most common base ata position where the base can nevertheless vary; (2) upper case lettersindicate highly-conserved bases, i.e. the ‘AUGG’ sequence is constant orrarely, if ever, changes, with the exception being the IUPAC ambiguitycode ‘R’ which indicates that a purine (adenine or guanine) is normallyobserved at this position; and (3) the sequence in brackets ((gee)) isof uncertain significance.

In one non-limiting embodiment, the IL2/IL2Rα fusion protein comprisesan IL2 leader optimized Kozak sequence as set forth in SEQ ID NO: 92(gccaccATGGACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT) ora functional variant or fragment thereof. A functional variant orfragment of a Kozak sequence will retain the ability to increasetranslation of the protein when compared to the level of translationfrom a sequence lacking the leader. Such a functional fragment cancomprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 30, 40 continuous nucleotides of a Kozak sequence or the sequenceset forth in SEQ ID NO:92 or SEQ ID NO:99 (gccaccATGG). Alternatively, afunctional variant can comprise at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the Kozak sequenceor the sequence set forth in SEQ ID NO:92 or SEQ ID NO:99.

7.5 Cells and Vectors

In certain aspects, provided herein are cells (e.g., host cells)expressing (e.g., recombinantly) fusion proteins described herein andexpression vectors comprising nucleotides that encode fusion proteinsdescribed herein. Provided herein are vectors (e.g., expression vectors)comprising polynucleotides comprising nucleotide sequences encoding afusion protein for recombinant expression in host cells.

In some embodiments, the host cell comprises the nucleic acids describedherein.

In some embodiments, the host cell is a eukaryotic cell. In someembodiments, the host cell is selected from the group consisting of amammalian cell, an insect cell, a yeast cell, a transgenic mammaliancell, and a plant cell. In some embodiments, the host cell is aprokaryotic cell. In some embodiments, the prokaryotic cell is abacterial cell.

In some embodiments, the host cell is a mammalian cell. Such mammalianhost cells include but are not limited to CHO, VERO, BHK, HeLa, MDCK,HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (amurine myeloma cell line that does not endogenously produce anyimmunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO,HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20,BMT10 and HsS78Bst cells.

As used herein, an expression vector refers to any nucleic acidconstruct which contains the necessary elements for the transcriptionand translation of an inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation,when introduced into an appropriate host cell. Expression vectors caninclude plasmids, phagemids, viruses, and derivatives thereof.

A gene expression control sequence as used herein is any regulatorynucleotide sequence, such as a promoter sequence or promoter-enhancercombination, which facilitates the efficient transcription andtranslation of the coding nucleic acid to which it is operably linked.The gene expression control sequence may, for example, be a mammalian orviral promoter, such as a constitutive or inducible promoter.Constitutive mammalian promoters include, but are not limited to, thepromoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actinpromoter, and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40),papilloma virus, adenovirus, human immunodeficiency virus (HIV), Roussarcoma virus, cytomegalovirus, the long terminal repeats (LTR) ofMoloney leukemia virus, and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as geneexpression sequences of the disclosure also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

For the purposes of this disclosure, numerous expression vector systemscan be employed. These expression vectors are typically replicable inthe host organisms either as episomes or as an integral part of the hostchromosomal DNA. Expression vectors can include expression controlsequences including, but not limited to, promoters (e.g.,naturally-associated or heterologous promoters), enhancers, signalsequences, splice signals, enhancer elements, and transcriptiontermination sequences. Preferably, the expression control sequences areeukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells. Expression vectors can also utilizeDNA elements which are derived from animal viruses such as bovinepapilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus,retroviruses (RSV, MMTV or MOMLV), cytomegalovirus (CMV), or SV40 virus.Others involve the use of polycistronic systems with internal ribosomebinding sites.

Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No.4,704,362). Cells which have integrated the DNA into their chromosomescan be selected by introducing one or more markers which allow selectionof transfected host cells. The marker can provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation.

An example of a vector useful for optimized expression of the fusionproteins used in the methods of the present disclosure is NEOSPLA (U.S.Pat. No. 6,159,730). This vector contains the cytomegaloviruspromoter/enhancer, the mouse beta globin major promoter, the SV40 originof replication, the bovine growth hormone polyadenylation sequence,neomycin phosphotransferase exon 1 and exon 2, the dihydrofolatereductase gene and leader sequence. Vector systems are also taught inU.S. Pat. Nos. 5,736,137 and 5,658,570. This system provides for highexpression levels, e.g., >30 pg/cell/day. Other exemplary vector systemsare disclosed e.g., in U.S. Pat. No. 6,413,777.

In other embodiments the polypeptides of the instant disclosure areexpressed using polycistronic constructs. In these expression systems,multiple gene products of interest such as multiple polypeptides ofmultimer binding protein can be produced from a single polycistronicconstruct. These systems advantageously use an internal ribosome entrysite (IRES) to provide relatively high levels of polypeptides ineukaryotic host cells. Compatible IRES sequences are disclosed in U.S.Pat. No. 6,193,980.

More generally, once the vector or DNA sequence encoding a polypeptidehas been prepared, the expression vector can be introduced into anappropriate host cell. That is, the host cells can be transformed.Introduction of the plasmid into the host cell can be accomplished byvarious techniques well known to those of skill in the art, as discussedabove. The transformed cells are grown under conditions appropriate forthe production of the fusion protein, and assayed for fusion proteinsynthesis. Exemplary assay techniques include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS),immunohistochemistry, and the like.

7.6 Pharmaceutical Compositions

The various IL2/IL2Rα fusion proteins disclosed herein (also referred toherein as “active compounds”) can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the fusion protein and a pharmaceutically acceptable carrier.As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

In some embodiments, disclosed is a pharmaceutical compositioncomprising (a) a IL2/IL2Rα fusion protein as described herein and (b) apharmaceutically acceptable excipient.

In some embodiments, disclosed is a pharmaceutical compositioncomprising (a) a composition comprising a IL2/IL2Rα fusion protein asdescribed herein and (b) a pharmaceutically acceptable excipient.

In some embodiments, disclosed is a pharmaceutical compositioncomprising (a) a nucleic acid as described herein and (b) apharmaceutically acceptable excipient.

In some embodiments, disclosed is a pharmaceutical compositioncomprising (a) a vector as described herein and (b) a pharmaceuticallyacceptable excipient.

In some embodiments, disclosed is a pharmaceutical compositioncomprising (a) a host cell as described herein and (b) apharmaceutically acceptable excipient.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal. In addition, it may be desirable to administer atherapeutically effective amount of the pharmaceutical compositionlocally to an area in need of treatment. This can be achieved by, forexample, local or regional infusion or perfusion during surgery, topicalapplication, injection, catheter, suppository, or implant (for example,implants formed from porous, non-porous, or gelatinous materials,including membranes, such as sialastic membranes or fibers), and thelike. In another embodiment, the therapeutically effective amount of thepharmaceutical composition is delivered in a vesicle, such as liposomes(see, e.g., Langer, Science 249:1527-33, 1990 and Treat et al., inLiposomes in the Therapy of Infectious Disease and Cancer, LopezBerestein and Fidler (eds.), Liss, N.Y., pp. 353-65, 1989).

In yet another embodiment, the therapeutically effective amount of thepharmaceutical composition can be delivered in a controlled releasesystem. In one example, a pump can be used (see, e.g., Langer, Science249:1527-33, 1990; Sefton, Crit. Rev. Biomed. Eng. 14:201-40, 1987;Buchwald et al., Surgery 88:507-16, 1980; Saudek et al., N Engl. J Med.321:574-79, 1989). In another example, polymeric materials can be used(see, e.g., Levy et al., Science 228:190-92, 1985; During et al., Ann.Neural. 25:351-56, 1989; Howard et al., J Neurosurg. 71:105-12, 1989).Other controlled release systems, such as those discussed by Langer(Science 249:1527-33, 1990), can also be used.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELS (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressurized container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Systemic administration can also be by transmucosal ortransdermal means.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art. The compounds can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensionscan also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such a functional compound for thetreatment of individuals. The pharmaceutical compositions can beincluded in a container, pack, or dispenser together with instructionsfor administration.

The effective amount of an IL2/IL2Rα fusion protein useful formodulating such functions will depend on the subject being treated, theseverity of the affliction, and the manner of administration of theIL2/IL2Rα fusion protein. Exemplary doses include about 104 to about 107IU of IL2 activity per adult, about 104 to 105 IU of IL2 activity peradult, about 105 to about 106 IU of IL2 activity per adult, about 106 toabout 107 IU of IL2 activity per adult. In other instances, thetherapeutically effective dose of the IL2/IL2Rα fusion protein is about105 IU of IL2 activity±100-fold, is about 105 IU of IL2activity±10-fold, about 105 IU of IL2 activity±2-fold, about 105 IU ofIL2 activity±20-fold, about 105 IU of IL2 activity±30-fold, about 105 IUof IL2 activity±40-fold, about 105 IU of IL2 activity±50-fold, about 105IU of IL2 activity±60-fold, about 105 IU of IL2 activity±70-fold, about105 IU of IL2 activity±80-fold, or about 105 IU of IL2 activity±90-fold.In a specific non-limiting embodiment, a human IL2 fusion protein isadministered at this dosage.

7.7 Uses and Methods 7.7.1 Methods of Making Compositions DisclosedHerein

As discussed above, the IL2/IL2Rα fusion proteins disclosed herein canbe used to create new IL2/IL2Rα fusion proteins by modifying IL2, IL2Rα,or any heterologous moiety sequence described herein. Thus, in anotheraspect described herein, the structural features of an IL2/IL2Rα fusionprotein described herein are used to create structurally relatedIL2/IL2Rα fusion proteins that retain at least one functional propertyof the IL2/IL2Rα fusion protein described herein, such as binding tohuman IL2Rα and cynomolgus IL2Rα. For example, one or more of IL2,IL2Rα, or any heterologous moiety sequence described herein can becombined recombinantly with known framework regions and/or otherproteins to create additional, recombinantly-engineered, fusion proteindescribed herein, as discussed above.

In some embodiments, disclosed herein are methods of producing anIL2/IL2Rα fusion protein comprising culturing a host cell comprising thefusion protein under suitable conditions and recovering the fusionprotein. In some embodiments, the host cell is a eukaryotic cell or aprokaryotic cell. In some embodiments, the host cell is a mammaliancell, an insect cell, a fungal cell, a plant cell, a transgenicmammalian cell, or a bacterial cell. In some embodiments, the host cellis selected from the group consisting of a CHO cell, a HEK 293 cell, aNS0 cell, a Per C6 cell, a BHK cell, and a COS cell. In one embodiment,the host cell is a bacterial cell. In a particular embodiment, thebacterial cell is Escherichia coli.

Other types of modifications include those described in the previoussection. The starting material for the engineering method is one or moreof the IL2 or IL2Rα sequences provided herein. To create the engineeredfusion protein, it is not necessary to actually prepare (i.e., expressas a protein) a fusion protein having one or more of the IL2 or IL2Rαsequences provided herein. Rather, the information contained in thesequence(s) is used as the starting material to create a “secondgeneration” sequence(s) derived from the original sequence(s) and thenthe “second generation” sequence(s) is prepared and expressed as aprotein.

Accordingly, provided herein are methods for preparing a fusion proteincomprising: (a) providing IL2 and IL2Rα; (b) altering at least one aminoacid residue within IL2 and IL2Rα to create at least one altered fusionprotein sequence; and (c) expressing the altered fusion protein sequenceas a protein.

Also provided herein are methods for preparing a fusion proteincomprising: (a) providing IL2 and IL2Rα; (b) altering at least oneglycosylation within IL2 and IL2Rα to create at least one alteredglycosylation site; and (c) expressing the altered fusion proteinsequence as a protein.

The altered antibody can exhibit one or more, two or more, three ormore, four or more, five or more, six or more, seven or more, eight ormore, nine or more, or all of the functional properties set forth as (1)through (10) above. The functional properties of the altered antibodiescan be assessed using standard assays available in the art.

In some embodiments of the methods of engineering the IL2/IL2Rα fusionproteins described herein, mutations can be introduced randomly orselectively along all or part of an IL2/IL2Rα fusion protein codingsequence and the resulting modified IL2/IL2Rα fusion proteins can bescreened for binding activity and/or other functional properties asdescribed herein. Mutational methods have been described in the art. Forexample, PCT Publication WO 02/092780 by Short describes methods forcreating and screening mutations using saturation mutagenesis, syntheticligation assembly, or a combination thereof. Alternatively, PCTPublication WO 03/074679 by Lazar et al. describes methods of usingcomputational screening methods to optimize physiochemical properties ofproteins.

Compositions further include isolated polynucleotides that encode thevarious fusion proteins described herein above, and variants andfragments thereof. Vectors and expression cassettes comprising thepolynucleotides described herein are further disclosed. Expressioncassettes will generally include a promoter operably linked to apolynucleotide and a transcriptional and translational terminationregion.

The use of the term “polynucleotide” is not intended to limit thepresent disclosure to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides, can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues.

In constructs that include more than one processing or cleavage site, itwill be understood that such sites can be the same or different.

An “isolated” or “purified” polynucleotide or protein, or biologicallyactive portion thereof, is substantially or essentially free fromcomponents that normally accompany or interact with the polynucleotideor protein as found in its naturally occurring environment. Thus, anisolated or purified polynucleotide or protein is substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Optimally, an “isolated”polynucleotide is free of sequences (optimally protein encodingsequences) that naturally flank the polynucleotide (i.e., sequenceslocated at the 5′ and 3′ ends of the polynucleotide) in the genomic DNAof the organism from which the polynucleotide is derived. For example,in various embodiments, the isolated polynucleotide can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequence that naturally flank the polynucleotide in genomic DNA of thecell from which the polynucleotide is derived.

A protein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, 5%, or 1%(by dry weight) of contaminating protein. When the protein of thedisclosure or biologically active portion thereof is recombinantlyproduced, optimally culture medium represents less than about 30%, 20%,10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Conventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art may be employed herein. Suchtechniques are explained fully in the literature. See, e.g., Sambrook etal., “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocolsin Molecular Biology” Volumes I-III [Ausubel, R. M., ed. (1994)]; “CellBiology: A Laboratory Handbook” Volumes I-III [J. E. Celis, ed.(1994))]; “Current Protocols in Immunology” Volumes I-III [Coligan, J.E., ed. (1994)]; “Oligonucleotide Synthesis” (M. J. Gaited. 1984);“Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)];“Transcription And Translation” [B. D. Hames & S. J. Higgins, eds.(1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)];“Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “APractical Guide To Molecular Cloning” (1984).

A vector which comprises the above-described polynucleotides operablylinked to a promoter is also provided herein. A nucleotide sequence is“operably linked” to an expression control sequence (e.g., a promoter)when the expression control sequence controls and regulates thetranscription and translation of that sequence. The term “operablylinked” when referring to a nucleotide sequence includes having anappropriate start signal (e.g., ATG) in front of the nucleotide sequenceto be expressed and maintaining the correct reading frame to permitexpression of the sequence under the control of the expression controlsequence and production of the desired product encoded by the sequence.If a gene that one desires to insert into a recombinant nucleic acidmolecule does not contain an appropriate start signal, such a startsignal can be inserted in front of the gene. A “vector” is a replicon,such as plasmid, phage or cosmid, to which another nucleic acid segmentmay be attached so as to bring about the replication of the attachedsegment. The promoter may be, or is identical to, a bacterial, yeast,insect or mammalian promoter. Further, the vector may be a plasmid,cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryoticviral DNA. Other numerous vector backbones known in the art as usefulfor expressing protein may be employed. Such vectors include, but arenot limited to: adenovirus, simian virus 40 (SV40), cytomegalovirus(CMV), mouse mammary tumor virus (MMTV), Moloney murine leukemia virus,DNA delivery systems, i.e. liposomes, and expression plasmid deliverysystems. Further, one class of vectors comprises DNA elements derivedfrom viruses such as bovine papilloma virus, polyoma virus, baculovirus,retroviruses or Semliki Forest virus. Such vectors may be obtainedcommercially or assembled from the sequences described by methodswell-known in the art.

A host vector system for the production of a polypeptide which comprisesthe vector of a suitable host cell is provided herein. Suitable hostcells include, but are not limited to, prokaryotic or eukaryotic cells,e.g. bacterial cells (including gram positive cells), yeast cells,fungal cells, insect cells, and animal cells. Numerous mammalian cellsmay be used as hosts, including, but not limited to, the mousefibroblast cell NIH 3T3, CHO cells, HeLa cells, Ltk cells, etc.Additional animal cells, such as R1.1, B-W and L-M cells, African GreenMonkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insectcells (e.g., Sf9), and human cells and plant cells in tissue culture canalso be used.

A wide variety of host/expression vector combinations may be employed inexpressing the polynucleotide sequences presented herein. Usefulexpression vectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage A, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2!l plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences (sequences thatcontrol the expression of a nucleotide sequence operably linked to it)may be used in these vectors to express the polynucleotide sequencesprovided herein. Such useful expression control sequences include, forexample, the early or late promoters of SV40, CMV, vaccinia, polyoma oradenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the LTR system, the major operator and promoter regions of phageA, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase (e.g., Pho5), the promoters of the yeast a-matingfactors, and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the polynucleotidesequences provided herein. Neither will all hosts function equally wellwith the same expression system. However, one skilled in the art will beable to select the proper vectors, expression control sequences, andhosts without undue experimentation to accomplish the desired expressionwithout departing from the scope of this disclosure. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular nucleotide sequence or gene to be expressed, particularlyas regards potential secondary structures. Suitable unicellular hostswill be selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the nucleotide sequencesto be expressed, and the ease of purification of the expressionproducts.

In preparing the expression cassette, the various polynucleotides may bemanipulated, so as to provide for the polynucleotide sequences in theproper orientation and, as appropriate, in the proper reading frame.Toward this end, adapters or linkers may be employed to join thepolynucleotides or other manipulations may be involved to provide forconvenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For example, linkers such as twoglycines may be added between polypeptides. Methionine residues encodedby ATG nucleotide sequences may be added to allow initiation of genetranscription. For this purpose, in vitro mutagenesis, primer repair,restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Further provided is a method of producing a polypeptide which comprisesexpressing a polynucleotide encoding a fusion protein disclosed hereinin a host cell under suitable conditions permitting the production ofthe polypeptide and recovering the polypeptide so produced.

7.7.2 Therapeutic Uses and Methods

The fusion proteins and methods described herein have numerous in vitroand in vivo utilities involving, for example, enhancement of immuneresponse, such as by inhibiting (or antagonizing) IL2Rα (e.g.,signaling), or detection of IL2. For example, the IL2/IL2Rα fusionproteins described herein can be administered to cells in culture, invitro or ex vivo, or to human subjects, e.g., in vivo, to enhanceimmunity in a variety of diseases. Accordingly, provided herein aremethod of treating a disease or disorder a subject in need thereof,comprising administering to the subject an IL2/IL2Rα fusion proteindescribed herein such that the immune response in the subject ismodified. In some embodiments, the response is enhanced, stimulated orup-regulated.

Subjects suitable for the present methods include human patients in whomenhancement of an immune response would be desirable. The methods areparticularly suitable for treating human patients having a disorder thatcan be treated by augmenting an immune response (e.g., a T-cell mediatedimmune response, e.g., an antigen specific T cell response). In someembodiments, the methods are particularly suitable for treatment ofcancer in vivo. To achieve antigen-specific enhancement of immunity,IL2/IL2Rα fusion proteins described herein can be administered togetherwith an antigen of interest or the antigen can already be present in thesubject to be treated (e.g., a tumor-bearing or virus-bearing subject).When the IL2/IL2Rα fusion proteins described herein are administeredtogether with another agent, the two can be administered separately orsimultaneously.

Also encompassed are methods for detecting the presence of human IL2 orhuman IL2Rα in a sample, or measuring the amount of human IL2 antigen,comprising contacting the sample, and a control sample, with a fusionprotein or monoclonal antibody, e.g., a human monoclonal antibody, or anantigen binding portion thereof, which specifically binds to human IL2or IL2Rα, under conditions that allow for formation of a complex betweenthe IL2/IL2Rα fusion proteins described herein and human IL2Rα. Theformation of a complex is then detected, wherein a difference complexformation between the sample compared to the control sample isindicative the presence of human IL2 or IL2Rα in the sample. Moreover,the IL2/IL2Rα fusion proteins described herein can be used to purifyhuman IL2 or IL2Rα via immunoaffinity purification.

Given the ability of IL2/IL2Rα fusion proteins described herein tostimulate or co-stimulate T cell responses, e.g., antigen-specific Tcell responses, such as by inhibiting negative effects of IL2 or IL2Rα,provided herein are in vitro and in vivo methods of using the IL2/IL2Rαfusion proteins described herein to stimulate, enhance or upregulateantigen-specific T cell responses, e.g., anti-tumor T cell responses. Insome embodiments, CD3 stimulation is also provided (e.g., bycoincubation with a cell expressing membrane CD3), which stimulation canbe provided at the same time, before, or after stimulation with anIL2/IL2Rα fusion proteins described herein. For example, provided hereinare methods of stimulating an antigen-specific T cell responsecomprising contacting said T cell with an IL2/IL2Rα fusion proteinsdescribed herein, and optionally with an anti-CD3 antibody, such that anantigen-specific T cell response is stimulated.

Any suitable indicator of an antigen-specific T cell response can beused to measure the antigen-specific T cell response. Non-limitingexamples of such suitable indicators include increased T cellproliferation in the presence of the antibody and/or increase cytokineproduction in the presence of the antibody. In some embodiments,interleukin-2 and/or interferon-γ production by the antigen-specific Tcell is stimulated.

T cells that can be enhanced or co-stimulated with IL2/IL2Rα fusionproteins described herein include CD4⁺ T cells and CD8⁺ T cells. The Tcells can be Teff cells, e.g., CD4⁺ Teff cells, CD8⁺ Teff cells, Thelper(Th) cells (e.g., Th1 cells) or T cytotoxic (Tc) cells.

Further encompassed are methods of stimulating an immune response (e.g.,an antigen-specific T cell response) in a subject comprisingadministering an IL2/IL2Rα fusion protein described herein to thesubject such that an immune response (e.g., an antigen-specific T cellresponse) in the subject is stimulated. In some embodiments, the subjectis a tumor-bearing subject and an immune response against the tumor isstimulated. A tumor can be a solid tumor or a liquid tumor, e.g., ahematological malignancy. In some embodiments, a tumor is an immunogenictumor. In some embodiments, a tumor is non-immunogenic. In someembodiments, a tumor is PD-L1 positive. In some embodiments a tumor isPD-L1 negative. A subject can also be a virus-bearing subject and animmune response against the virus is stimulated.

Further provided are methods for inhibiting growth of tumor cells in asubject comprising administering to the subject an IL2/IL2Rα fusionprotein described herein such that growth of the tumor is inhibited inthe subject. Also provided are methods of treating a viral infection ina subject comprising administering to the subject an IL2/IL2Rα fusionprotein described herein such that the viral infection is treated in thesubject.

In some embodiments, an IL2/IL2Rα fusion protein described herein isgiven to a subject as an adjunctive therapy. Treatments of subjectshaving cancer with an IL2/IL2Rα fusion protein described herein can leadto prolonged survival, e.g., long-term durable response relative to thecurrent standard of care; long term survival of at least 3 months, 6months, 9 months, 1, 2, 3, 4, 5, 10 or more years, or recurrence-freesurvival of at least 3 months, 6 months, 9 months, 1, 2, 3, 4, 5, or 10or more years. In some embodiments, treatment of a subject having cancerwith an IL2/IL2Rα fusion proteins described herein prevents recurrenceof cancer or delays recurrence of cancer by, e.g., 3 months, 6 months, 9months, 1, 2, 3, 4, 5, or 10 or more years. An IL2/IL2Rα fusion proteintreatment can be used as a first-, second-, or third-line treatment.

Treatment of a subject having cancer with an IL2/IL2Rα fusion proteindescribed herein can result in, e.g., stable disease, partial response,increased overall survival, increased disease free survival, or enhancedprogression free survival.

In some embodiments, an IL2/IL2Rα fusion protein described herein is notsignificantly toxic. For example, an IL2/IL2Rα fusion protein describedherein is not significantly toxic to an organ of a human, e.g., one ormore of the liver, kidney, brain, lungs, and heart, as determined, e.g.,in clinical trials. In some embodiments, an IL2/IL2Rα fusion proteindescribed herein does not significantly trigger an undesirable immuneresponse, e.g., autoimmunity or inflammation.

In some embodiments, treatment of a subject with an IL2/IL2Rα fusionprotein described herein does not result in overstimulation of theimmune system to the extent that the subject's immune system thenattacks the subject itself (e.g., autoimmune response) or results in,e.g., anaphylaxis. Thus, in some embodiments, the IL2/IL2Rα fusionproteins described herein do not cause anaphylaxis.

In some embodiments, treatment of a subject with an IL2/IL2Rα fusionprotein described herein does not cause significant inflammatoryreactions, e.g., immune-mediated pneumonitis, immune-mediated colitis,immune mediated hepatitis, immune-mediated nephritis or renaldysfunction, immune-mediated hypophysitis, immune-mediatedhypothyroidism and hyperthyroidism, or other immune-mediated adversereactions. In some embodiments, treatment of a subject with theIL2/IL2Rα fusion proteins described herein does not cause significantcardiac disorders, e.g., ventricular arrhythmia; eye disorders, e.g.,iridocyclitis; infusion-related reactions; increased amylase, increasedlipase; nervous system disorders, e.g., dizziness, peripheral andsensory neuropathy; skin and subcutaneous tissue disorders, e.g., rash,pruritus, exfoliative dermatitis, erythema multiforme, vitiligo orpsoriasis; respiratory, thoracic and mediastinal disorders, e.g., cough;fatigue; nausea; decreased appetite; constipation; arthralgia; ordiarrhea.

In some embodiments, the IL2/IL2Rα fusion proteins described hereinprovide synergistic anti-tumor effects in combination with anothercancer therapy, such as a compound that stimulates the immune system(e.g., an immuno-oncology agent), e.g., a compound described herein or acompound modulating a target described herein.

In some embodiments, the IL2/IL2Rα fusion proteins described herein isadministered via a topical, epidermal mucosal, intranasal, oral,vaginal, rectal, sublingual, topical, intravenous, intraperitoneal,intramuscular, intraarterial, intrathecal, intralymphatic,intralesional, intracapsular, intraorbital, intracardiac, intradermal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural or intrasternal route.

Various methods are provided for increasing the immune response in asubject. Such methods comprise administering to a subject in need of anincrease in the immune response a therapeutically effective amount of anIL2/IL2Rα fusion protein. As such, in specific embodiments, transientapplication of higher doses of IL2 are employed to boosted immuneeffector and memory responses.

Various methods are provided for decreasing the immune response in asubject. Such methods comprise administering to a subject in need of adecrease in the immune response a therapeutically effective amount of anIL2/IL2Rα fusion protein. There is much interest to harness thesuppressive power of Tregs to inhibit unwanted immune responses. Data inmouse and man shows that enhancing IL2R signaling with a low dose of IL2selectively boosts Tregs and enhances immune tolerogenic mechanisms.IL2/IL2Rα fusion proteins provided herein represent a new and improvedform of IL2 that more potentially enhances Tregs. Thus, the IL2/IL2Rαfusion proteins can be administered to patients with autoimmunediseases, chronic graft versus host disease, transplant rejectionreactions, and other conditions where the goal is to suppressself-reactivity.

These and other methods described herein are discussed in further detailbelow.

7.7.2.1 Cancer

In some embodiments, disclosed herein are methods of treating cancer.Inhibition of IL2Rα by an IL2/IL2Rα fusion protein can enhance theimmune response to cancerous cells in a patient having cancer. Providedherein are methods for treating a subject having cancer, comprisingadministering to the subject an IL2/IL2Rα fusion protein describedherein, such that the subject is treated, e.g., such that growth ofcancerous tumors is inhibited or reduced and/or that the tumors regressand/or that prolonged survival is achieved. An IL2/IL2Rα fusion proteincan be used alone to inhibit the growth of cancerous tumors.Alternatively, an IL2/IL2Rα fusion protein can be used in conjunctionwith another agent, e.g., another immunogenic agent, a standard cancertreatment, or another antibody, as described below.

Accordingly, provided herein are methods of treating cancer, e.g., byinhibiting growth of tumor cells, in a subject, comprising administeringto the subject a therapeutically effective amount of an IL2/IL2Rα fusionprotein disclosed herein. Cancers whose growth can be inhibited usingthe antibodies of the disclosure include cancers typically responsive toimmunotherapy and those that are not typically responsive toimmunotherapy. Cancers can be cancers with solid tumors or bloodmalignancies (liquid tumors). In some embodiments, the cancer is abladder cancer, breast cancer, uterine cancer, endometrial carcinoma,ovarian cancer, colorectal cancer, colon cancer, head and neck cancer,lung cancer, stomach cancer, germ cell cancer, bone cancer, squamouscell cancer, skin cancer, neoplasm of the central nervous system,lymphoma, leukemia, sarcoma, virus-related cancer, small-cell lungcancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin'sor non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma,cervical cancer, ovarian cancer, liver cancer, myeloma, salivary glandcarcinoma, kidney cancer, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, or head or neck cancer, and any combinations thereof.

Other non-limiting examples of cancers for treatment include squamouscell carcinoma, small-cell lung cancer, non-small cell lung cancer,squamous non-small cell lung cancer (NSCLC), nonsquamous NSCLC, glioma,gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma),ovarian cancer, liver cancer, colorectal cancer, endometrial cancer,kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g.,hormone refractory prostate adenocarcinoma), thyroid cancer,neuroblastoma, pancreatic cancer, glioblastoma (glioblastomamultiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma,breast cancer, colon carcinoma, and head and neck cancer (or carcinoma),gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal naturalkiller, melanoma (e.g., metastatic malignant melanoma, such as cutaneousor intraocular malignant melanoma), bone cancer, skin cancer, uterinecancer, cancer of the anal region, testicular cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,solid tumors of childhood, cancer of the ureter, carcinoma of the renalpelvis, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer,squamous cell cancer, T-cell lymphoma, environmentally-induced cancersincluding those induced by asbestos, virus-related cancers or cancers ofviral origin (e.g., human papilloma virus (HPV-related or -originatingtumors)), and hematologic malignancies derived from either of the twomajor blood cell lineages, i.e., the myeloid cell line (which producesgranulocytes, erythrocytes, thrombocytes, macrophages and mast cells) orlymphoid cell line (which produces B, T, NK and plasma cells), such asall types of leukemias, lymphomas, and myelomas, e.g., acute, chronic,lymphocytic and/or myelogenous leukemias, such as acute leukemia (ALL),acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL),and chronic myelogenous leukemia (CML), undifferentiated AML (MO),myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellmaturation), promyelocytic leukemia (M3 or M3 variant [M3V]),myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]),monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia(M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such asHodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B cellhematologic malignancy, e.g., B-cell lymphomas, T-cell lymphomas,lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma,mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1+) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle celllymphoma, angio immunoblastic T-cell lymphoma, angiocentric lymphoma,intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma,precursor T-lymphoblastic lymphoma, T-lymphoblastic; andlymphoma/leukaemia (T-Lbly/T-ALL), peripheral T-cell lymphoma,lymphoblastic lymphoma, post-transplantation lymphoproliferativedisorder, true histiocytic lymphoma, primary central nervous systemlymphoma, primary effusion lymphoma, B cell lymphoma, lymphoblasticlymphoma (LBL), hematopoietic tumors of lymphoid lineage, acutelymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt'slymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL),immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides orSezary syndrome), and lymphoplasmacytoid lymphoma (LPL) withWaldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, lightchain myeloma, nonsecretory myeloma, smoldering myeloma (also calledindolent myeloma), solitary plasmocytoma, and multiple myelomas, chroniclymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors ofmyeloid lineage, tumors of mesenchymal origin, including fibrosarcomaand rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the centraland peripheral nervous, including astrocytoma, schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer andteratocarcinoma, hematopoietic tumors of lymphoid lineage, for exampleT-cell and B-cell tumors, including but not limited to T-cell disorderssuch as T-prolymphocytic leukemia (T-PLL), including of the small celland cerebriform cell type; large granular lymphocyte leukemia (LGL) ofthe T-cell type; a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head orneck, renal cancer, rectal cancer, cancer of the thyroid gland; acutemyeloid lymphoma, as well as any combinations of said cancers. Themethods described herein can also be used for treatment of metastaticcancers, unresectable, refractory cancers (e.g., cancers refractory toprevious immunotherapy, e.g., with a blocking CTLA-4 or PD-1 antibody),and/or recurrent cancers.

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein isadministered to patients having a cancer that exhibited an inadequateresponse to, or progressed on, a prior treatment, e.g., a priortreatment with an immuno-oncology or immunotherapy drug, or patientshaving a cancer that is refractory or resistant, either intrinsicallyrefractory or resistant (e.g., refractory to a PD-1 pathway antagonist),or a wherein the resistance or refractory state is acquired. Forexample, subjects who are not responsive or not sufficiently responsiveto a first therapy or who see disease progression following treatment,e.g., anti-PD-1 treatment, can be treated by administration of anIL2/IL2Rα fusion protein disclosed herein alone or in combination withanother therapy (e.g., with an anti-PD-1 therapy).

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein isadministered to patients who have not previously received (i.e., beentreated with) an immuno-oncology agent, e.g., a PD-1 pathway antagonist.

A method of treating a subject having cancer with an IL2/IL2Rα fusionprotein disclosed herein can comprise administering to a subject who hascancer cells that express IL2 or IL2Rα, a therapeutically effectiveamount of the IL2/IL2Rα fusion protein disclosed herein. Also providedherein are methods for predicting whether a subject will respond totreatment with an IL2/IL2Rα fusion protein disclosed herein, wherein themethods comprise determining the level of IL2 or IL2Rα in a patient, andif the subject is positive for IL2 or IL2Rα, then the subject is likelyto respond to a treatment with an IL2/IL2Rα fusion protein disclosedherein.

In some embodiments, a method of treating cancer in a subject comprisesfirst determining whether the subject is PD-L1 or PD-1 positive, e.g.,has tumor cells or TILs that express PD-L1 or PD-1, and if the subjecthas PD-L1 or PD-1 positive cancer or TIL cells, then administering tothe subject an IL2/IL2Rα fusion protein disclosed herein (and optionallya PD-1 or PD-L1 antagonist). A method of treating a subject havingcancer with an IL2/IL2Rα fusion protein disclosed herein (and optionallya PD-1 or PD-L1 antagonist) can comprise administering to a subject whohas cancer cells or TIL cells that express PD-L1 or PD-1, atherapeutically effective amount of an IL2/IL2Rα fusion proteindisclosed herein (and optionally a PD-1 or PD-L1 antagonist). Alsoprovided herein are methods for predicting whether a subject willrespond to treatment with an IL2/IL2Rα fusion protein disclosed herein(and optionally a PD-1 or PD-L1 antagonist), wherein the methodscomprise determining the level of PD-L1 or PD-1 in cancer or TIL cellsof the patient, and if cancer or TIL cells of the subject are PD-L1 orPD-1 positive, then the subject is likely to respond to a treatment withan IL2/IL2Rα fusion protein disclosed herein (and optionally a PD-1 orPD-L1 antagonist).

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein isadministered with a standard of care treatment. In some embodiments, anIL2/IL2Rα fusion protein disclosed herein is administered as amaintenance therapy, e.g., a therapy that is intended to prevent theoccurrence or recurrence of tumors.

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein isadministered with another treatment, e.g., radiation, surgery, orchemotherapy. For example, in some embodiments, adjunctive therapy usingan IL2/IL2Rα fusion protein disclosed herein is administered when thereis a risk that micrometastases can be present and/or in order to reducethe risk of a relapse.

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein isadministered as a monotherapy, or as the only immuno stimulatingtherapy. Antibodies to IL2Rα can also be combined with an immunogenicagent, such as cancerous cells, purified tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules), cells, andcells transfected with genes encoding immune stimulating cytokines (Heet al., (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumorvaccines that can be used include peptides of melanoma antigens, such aspeptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, ortumor cells transfected to express the cytokine GM-CSF (discussedfurther below).

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein iscombined with a vaccination protocol. Many experimental strategies forvaccination against tumors have been devised (see Rosenberg, S., 2000,Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62;Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D.2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCOEducational Book Spring: 730-738; see also Restifo, N. and Sznol, M.,Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997,Cancer: Principles and Practice of Oncology, Fifth Edition). In one ofthese strategies, a vaccine is prepared using autologous or allogeneictumor cells. These cellular vaccines have been shown to be mosteffective when the tumor cells are transduced to express GM-CSF. GM-CSFhas been shown to be a potent activator of antigen presentation fortumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A.90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. In some embodiments, the IL2/IL2Rα fusionprotein disclosed herein is used in conjunction with a collection ofrecombinant proteins and/or peptides expressed in a tumor in order togenerate an immune response to these proteins. These proteins arenormally viewed by the immune system as self-antigens and are thereforetolerant to them. The tumor antigen can include the protein telomerase,which is required for the synthesis of telomeres of chromosomes andwhich is expressed in more than 85% of human cancers and in only alimited number of somatic tissues (Kim et al. (1994) Science 266:2011-2013). Tumor antigen can also be “neo-antigens” expressed in cancercells because of somatic mutations that alter protein sequence or createfusion proteins between two unrelated sequences (i.e., bcr-abl in thePhiladelphia chromosome), or idiotype from B cell tumors.

Other tumor vaccines can include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which can be used in conjunction with the fusionprotein of the present disclosure is purified heat shock proteins (HSP)isolated from the tumor tissue itself. These heat shock proteins containfragments of proteins from the tumor cells and these HSPs are highlyefficient at delivery to antigen presenting cells for eliciting tumorimmunity (Suot & Srivastava (1995) Science 269: 1585-1588; Tamura et al.(1997) Science 278: 117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DCs can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withan IL2/IL2Rα fusion protein disclosed herein to activate more potentanti-tumor responses.

In some embodiments, methods of treatment of cancer using an IL2/IL2Rαfusion protein disclosed herein are combined with standard cancertreatments (e.g., surgery, radiation, and chemotherapy). An example ofsuch a combination is an anti-TIM3 antibody in combination with anIL2/IL2Rα fusion protein disclosed herein for the treatment of melanoma.The scientific rationale behind the combined use of an IL2/IL2Rα fusionprotein disclosed herein and chemotherapy is that cell death, that is aconsequence of the cytotoxic action of most chemotherapeutic compounds,should result in increased levels of tumor antigen in the antigenpresentation pathway. Other combination therapies that can result insynergy with an IL2/IL2Rα fusion protein disclosed herein through celldeath are radiation, surgery, and hormone deprivation. Each of theseprotocols creates a source of tumor antigen in the host. Angiogenesisinhibitors can also be combined with an IL2/IL2Rα fusion proteindisclosed herein. Inhibition of angiogenesis leads to tumor cell deathwhich can feed tumor antigen into host antigen presentation pathways.

The IL2/IL2Rα fusion protein disclosed herein described herein can alsobe used in combination with bispecific antibodies that target Fcα or Fcγreceptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used totarget two separate antigens. For example anti-Fc receptor/anti-tumorantigen (e.g., Her-2/neu) bispecific antibodies have been used to targetmacrophages to sites of tumor. This targeting can more effectivelyactivate tumor specific responses. Alternatively, antigen can bedelivered directly to DCs by the use of bispecific antibodies which bindto tumor antigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms can be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-β (Kehrl et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365).Antibodies to each of these entities can be used in combination with anIL2/IL2Rα fusion protein disclosed herein to counteract the effects ofthe immunosuppressive agent and favor tumor immune responses by thehost.

Other antibodies which activate host immune responsiveness can be usedin combination with an IL2/IL2Rα fusion protein disclosed herein. Theseinclude molecules on the surface of dendritic cells which activate DCfunction and antigen presentation. Anti-CD40 antibodies are able tosubstitute effectively for T cell helper activity (Ridge et al. (1998)Nature 393: 474-478) and can be used in conjunction with an IL2/IL2Rαfusion protein disclosed herein. Activating antibodies to T cellcostimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097),OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero etal. (1997) Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff et al.(1999) Nature 397: 262-266) can also provide for increased levels of Tcell activation. Inhibitors of PD1 or PD-L1 can also be used inconjunction with an IL2/IL2Rα fusion protein disclosed herein. Othercombinations are provided elsewhere herein.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit can be obtained fromgraft vs. tumor responses. In some embodiments, an IL2/IL2Rα fusionprotein disclosed herein is used to increase the effectiveness of thedonor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to stimulateantigen-specific T cells against tumor (Greenberg & Riddell (1999)Science 285: 546-51). These methods can also be used to activate T cellresponses to infectious agents such as CMV. In some embodiments, ex vivoactivation in the presence of an IL2/IL2Rα fusion protein disclosedherein can increase the frequency and activity of the adoptivelytransferred T cells.

7.7.2.2 Inflammatory Disease or Autoimmune Disease

In some embodiments, disclosed herein are methods of treating a diseaseor disorder a subject in need thereof, wherein the disease or disorderis an inflammatory disease or an autoimmune disease. In someembodiments, the methods comprise administering to the subject atherapeutically effective amount of an IL2/IL2Rα fusion proteindisclosed herein.

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein isadministered to patients having an inflammatory disease or an autoimmunedisease that exhibited an inadequate response to, or progressed on, aprior treatment. In some embodiments, an IL2/IL2Rα fusion proteindisclosed herein is administered to patients who have not previouslyreceived (i.e., been treated with) treatment for the an inflammatorydisease or an autoimmune disease.

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein isadministered with a standard of care treatment for an inflammatorydisease or an autoimmune disease. In some embodiments, an IL2/IL2Rαfusion protein disclosed herein is administered as a maintenance therapyfor an inflammatory disease or an autoimmune disease, e.g., a therapythat is intended to prevent the occurrence or recurrence ofinflammation.

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein isadministered as a monotherapy for treatment of an inflammatory diseaseor an autoimmune disease, or as the only immuno stimulating therapy fortreatment of an inflammatory disease or an autoimmune disease. In someembodiments, an IL2/IL2Rα fusion protein disclosed herein is combinedwith a vaccination protocol for treatment of an inflammatory disease oran autoimmune disease. In some embodiments, an IL2/IL2Rα fusion proteindisclosed herein is combined with an antibody used for treatment of aninflammatory disease or an autoimmune disease.

In some embodiments, the inflammatory disease or an autoimmune diseaseis selected from the group consisting of type 1 diabetes, multiplesclerosis, rheumatoid arthritis, celiac disease, systemic lupuserythematous, lupus nephritis, cutaneous lupus, juvenile idiopathicarthritis, Crohn's disease, ulcerative colitis or systemic sclerosis,graft versus host disease, psoriasis, alopecia areata, HCV-inducedvasculitis, Sjogren's syndrome, Pemphigus, Ankylosing Spondylitis,Behcet's Disease, Wegener's Granulomatosis, Takayasu's Disease,Autoimmune Hepatitis, Sclerosing Cholangitis, Gougerot-sjögren, andMacrophage Activation Syndrome.

7.7.2.3 Infectious Disease

Methods described herein can also be used to treat patients that havebeen exposed to particular toxins or pathogens. Accordingly, in someembodiments, disclosed herein are methods of treating a disease ordisorder a subject in need thereof, wherein the disease or disorder isan infectious disease. In some embodiments, the methods compriseadministering to the subject a therapeutically effective amount of anIL2/IL2Rα fusion protein disclosed herein to treat the infectiousdisease.

Similar to its application to tumors as discussed above, methodscomprising treatment with an IL2/IL2Rα fusion protein disclosed hereinare used alone, or as an adjuvant, in combination with vaccines, tostimulate the immune response to pathogens, toxins, and self-antigens.Examples of pathogens for which this therapeutic approach can beparticularly useful, include pathogens for which there is currently noeffective vaccine, or pathogens for which conventional vaccines are lessthan completely effective. These include, but are not limited to HIV,Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria, Leishmania,Staphylococcus aureus, Pseudomonas aeruginosa.

Some examples of pathogenic viruses causing infections treatable bymethods described herein include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods described herein include Chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella,Diphtheria, Salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods described herein include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods described herein include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, and Nippostrongylus brasiliensis.

In all of the above methods, treatment with an IL2/IL2Rα fusion proteindisclosed herein can be combined with other forms of immunotherapy,e.g., those described herein, such as cytokine treatment (e.g.,interferons, GM-CSF, G-CSF, IL2), or bispecific antibody therapy, whichprovides for enhanced presentation of tumor antigens (see, e.g, Holliger(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure2: 1121-1123).

7.7.2.4 Vaccines

The IL2/IL2Rα fusion proteins disclosed herein can be used to stimulateantigen-specific immune responses by co-administration of an IL2/IL2Rαfusion protein disclosed herein with an antigen of interest (e.g., avaccine). Accordingly, provided herein are methods of enhancing animmune response to an antigen in a subject, comprising administering tothe subject: (i) the antigen; and (ii) an IL2/IL2Rα fusion proteindisclosed herein, such that an immune response to the antigen in thesubject is enhanced. The antigen can be, for example, a tumor antigen, aviral antigen, a bacterial antigen or an antigen from a pathogen.Non-limiting examples of such antigens include those discussed in thesections above, such as the tumor antigens (or tumor vaccines) discussedabove, or antigens from the viruses, bacteria or other pathogensdescribed above.

In some embodiments, a peptide or fusion protein comprising the epitopeto which an IL2/IL2Rα fusion protein disclosed herein binds is used as avaccine instead of, or in addition to, an IL2/IL2Rα fusion proteindisclosed herein.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) described herein in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

7.7.2.5 Co-Administration with a Second Agent

As previously described, an IL2/IL2Rα fusion protein disclosed hereincan be co-administered with one or other more therapeutic agents, e.g.,a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. TheIL2/IL2Rα fusion protein disclosed herein can be linked to the agent (asan immuno-complex) or can be administered separate from the agent. Inthe latter case (separate administration), the IL2/IL2Rα fusion proteindisclosed herein can be administered before, after or concurrently withthe agent or can be co-administered with other known therapies, e.g., ananti-cancer therapy, e.g., radiation. Such therapeutic agents include,among others, anti-neoplastic agents such as doxorubicin (adriamycin),cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine andcyclophosphamide hydroxyurea which, by themselves, are only effective atlevels which are toxic or subtoxic to a patient. Cisplatin isintravenously administered as a 100 mg/ml dose once every four weeks andadriamycin is intravenously administered as a 60-75 mg/ml dose onceevery 21 days. Co-administration of an IL2/IL2Rα fusion proteindisclosed herein with chemotherapeutic agents provides two anti-canceragents which operate via different mechanisms which yield a cytotoxiceffect to human tumor cells. Such co-administration can solve problemsdue to development of resistance to drugs or a change in theantigenicity of the tumor cells which would render them unreactive withthe antibody.

Provided herein are methods of combination therapy in which an IL2/IL2Rαfusion protein disclosed herein is co-administered with one or moreadditional agents (a second therapeutic agent), e.g., small moleculedrugs, antibodies or antigen binding portions thereof, that areeffective in stimulating immune responses to thereby further enhance,stimulate or upregulate immune responses in a subject.

Generally, an IL2/IL2Rα fusion protein disclosed herein can be combinedwith (i) an agonist of a stimulatory (e.g., co-stimulatory) molecule(e.g., receptor or ligand) and/or (ii) an antagonist of an inhibitorysignal or molecule (e.g., receptor or ligand) on immune cells, such as Tcells, both of which result in amplifying immune responses, such asantigen-specific T cell responses. In some aspects, an immuno-oncologyagent is (i) an agonist of a stimulatory (including a co-stimulatory)molecule (e.g., receptor or ligand) or (ii) an antagonist of aninhibitory (including a co-inhibitory) molecule (e.g., receptor orligand) on cells, e.g., those inhibiting T cell activation or thoseinvolved in innate immunity, e.g., NK cells, and wherein theimmuno-oncology agent enhances innate immunity. Such immuno-oncologyagents are often referred to as immune checkpoint regulators, e.g.,immune checkpoint inhibitor or immune checkpoint stimulator.

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein isadministered with an agent that targets a stimulatory or inhibitorymolecule that is a member of the immunoglobulin super family (IgSF). Forexample, an IL2/IL2Rα fusion protein disclosed herein can beadministered to a subject with an agent that targets a member of theIgSF family to increase an immune response. For example, an IL2/IL2Rαfusion protein disclosed herein can be administered with an agent thattargets (or binds specifically to) a member of the B7 family ofmembrane-bound ligands that includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC(PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6 or aco-stimulatory or co-inhibitory receptor or ligand binding specificallyto a B7 family member.

An IL2/IL2Rα fusion protein disclosed herein can also be administeredwith an agent that targets a member of the TNF and TNFR family ofmolecules (ligands or receptors), such as CD40 and CD40L, OX-40, OX-40L,CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL/Apo2-L, TRAILR1/DR4,TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn 14, TWEAK,BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTpR, LIGHT, DcR3, HVEM,VEGI/TL1A, TRAMP/DR3, EDA1, EDA2, TNFR1, Lymphotoxin a/TNFp, TNFR2,TNFa, LTpR, Lymphotoxin a 1β2, FAS, FASL, RELT, DR6, TROY, and NGFR(see, e.g., Tansey (2009) Drug Discovery Today 00:1).

In some embodiments, an IL2/IL2Rα fusion protein disclosed herein isadministered with an agent comprising an anti-PD-1 antibody. Theanti-PD-1 antibody can be any antibody that binds PD-1 and inhibits theinteraction of PD-1 and PD-L1. In some embodiments, the anti-PD-1antibody is any anti-PD-1 antibody known in the art. In someembodiments, the second therapeutic agent comprises nivolumab. In someembodiments, the second therapeutic agent comprises pembrolizumab.

In some embodiments, wherein the second therapeutic agent comprises ananti-PD-L1 antibody. The anti-PD-L1 antibody can be any antibody thatbinds PD-L1 and inhibits the interaction of PD-1 and PD-L1. In someembodiments, the anti-PD-L1 antibody is any anti-PD-L1 antibody known inthe art. In some embodiments, the second therapeutic agent comprisesatezolizumab. In some embodiments, the second therapeutic agentcomprises durvalumab. In some embodiments, the second therapeutic agentcomprises avelumab.

In some embodiments, wherein the second therapeutic agent comprises ananti-CTLA-4 antibody. The anti-CTLA-4 antibody can be any antibody thatbinds CTLA-4 and inhibits its activity. In some embodiments, theanti-CTLA-4 antibody is any anti-CTLA-4 antibody known in the art. Insome embodiments, the second therapeutic agent comprises tremelimumab.In some embodiments, the second therapeutic agent comprises ipilimumab.

In some embodiments, wherein the second therapeutic agent comprises ananti-LAG3 antibody. The anti-LAG3 antibody can be any antibody thatbinds LAG-3 and inhibits its activity. In some embodiments, theanti-LAG3 antibody is any anti-LAG3 antibody known in the art. In someembodiments, the second therapeutic agent comprises 25F7.

In some embodiments, wherein the second therapeutic agent comprises ananti-CD137 antibody. The anti-CD137 antibody can be any antibody thatbinds CD137 and inhibits its activity. In some embodiments, theanti-CD137 antibody is any anti-CD137 antibody known in the art. In someembodiments, the second therapeutic agent comprises urelumab.

In some embodiments, wherein the second therapeutic agent comprises ananti-KIR antibody. The anti-KIR antibody can be any antibody that bindsKIR and inhibits its activity. In some embodiments, the anti-KIRantibody is any anti-KIR antibody known in the art. In some embodiments,the second therapeutic agent comprises lirilumab.

In some embodiments, wherein the second therapeutic agent comprises ananti-GITR antibody. The anti-GITR antibody can be any antibody thatbinds GITR and inhibits its activity. In some embodiments, the anti-GITRantibody is any anti-GITR antibody known in the art. In someembodiments, the second therapeutic agent comprises MK4166. In someembodiments, the second therapeutic agent comprises TRX518.

In other embodiments, the second therapy comprises administering ananti-TIM3 antibody. The anti-TIM3 antibody can be any antibody thatbinds TIM3 and inhibits its activity. In some embodiments, the anti-TIM3antibody is any anti-TIM3 antibody known in the art.

In certain embodiments, the second therapy comprises administering achemotherapeutic agent. In some embodiments, the chemotherapeutic agentis selected from a proteasome inhibitor, an immunomodulatory drug(IMiD), a Bet inhibitor, and any combination thereof. In someembodiments, the proteasome inhibitor is selected from bortezomib,ixazomib, carfilzomib, oprozomib and marizomib. In certain embodiments,the proteasome inhibitor comprises bortezomib.

In some embodiments, the second therapy comprises a radiotherapy. Anyradiotherapy known in the art can be used as the second therapy.

In some embodiments, the second therapy comprises administering an agentthat activates innate immune cells. In some embodiments, the agent thatactivates innate immune cells comprises an NLRP3 agonist. In someembodiments, the NLRP3 agonist comprises monosodium urate monohydrate(MSU) and/or the vaccine adjuvant alum. In some embodiments, the agentthat activates innate immune cells is a toll like receptor 7 (TLR7)agonist. In some embodiments, the TLR7 agonist comprises imiquimod(R837), GS-9620 (see Tsai et al., J. Virology doi:10.1128/JVI.02166-16(Feb. 8, 2017)), ORN R-2336 (Miltenyl Biotec), or any combinationthereof.

In some embodiments, the second therapy comprises administering an agentthat enhances the survival of natural killer (NK) cells, CD8⁺ T cells,or both.

In certain embodiments, the second therapy comprises administering anagent selected from the group consisting of doxorubicin (ADRIAMYCIN®),cisplatin, carboplatin, bleomycin sulfate, carmustine, chlorambucil(LEUKERAN®), cyclophosphamide (CYTOXAN®; NEOSAR®), lenalidomide(REVLIMID®), bortezomib (VELCADE®), dexamethasone, mitoxantrone,etoposide, cytarabine, bendamustine (TREANDA®), rituximab (RITUXAN®),ifosfamide, vincristine (ONCOVIN®), fludarabine (FLUDARA®), thalidomide(THALOMID®), alemtuzumab (CAMPATH®), ofatumumab (ARZERRA®), everolimus(AFINITOR®, ZORTRESS®), carfilzomib (KYPROLIS™), and any combinationthereof.

Exemplary agents that modulate one of the above proteins and can becombined with the fusion protein described herein, for treating cancer,include: YERVOY® (ipilimumab) or Tremelimumab (to CTLA-4), galiximab (toB7.1), BMS-936558 (to PD-1), MK-3475 (to PD-1), atezolizumab(TECENTRIQ®), AMP224 (to B7DC), BMS-936559 (to B7-H1), MPDL3280A (toB7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (to B7H3), IMP321(to LAG-3), BMS-663513 (to CD137), PF-05082566 (to CD137), CDX-1127 (toCD27), anti-OX40 (Providence Health Services), huMAbOX40L (to OX40L),Atacicept (to TACI), CP-870893 (to CD40), Lucatumumab (to CD40),Dacetuzumab (to CD40), Muromonab-CD3 (to CD3); anti-GITR antibodiesMK4166, TRX518, Medi1873, INBRX-110, LK2-145, GWN-323, GITRL-Fc, or anycombination thereof.

Other molecules that can be combined with the fusion protein for thetreatment of a disease or disorder include antagonists of inhibitoryreceptors on NK cells or agonists of activating receptors on NK cells.For example, the fusion proteins described herein can be combined withantagonists of KIR (e.g., lirilumab).

T cell activation is also regulated by soluble cytokines, and the fusionproteins described herein can be administered to a subject, e.g., havingcancer, with antagonists of cytokines that inhibit T cell activation oragonists of cytokines that stimulate T cell activation.

In some embodiments, the fusion proteins described herein can be used incombination with (i) antagonists (or inhibitors or blocking agents) ofproteins of the IgSF family or B7 family or the TNF family that inhibitT cell activation or antagonists of cytokines that inhibit T cellactivation (e.g., IL-6, IL-10, TGF-β, VEGF; “immunosuppressivecytokines”) and/or (ii) agonists of stimulatory receptors of the IgSFfamily, B7 family or the TNF family or of cytokines that stimulate Tcell activation, for stimulating an immune response, e.g., for treatingproliferative diseases, such as cancer.

Yet other agents for combination therapies include agents that inhibitor deplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155(WO11/70024, WO11/107553, WO11/131407, W013/87699, W013/119716,WO13/132044) or FPA-008 (WO11/140249; W013169264; WO14/036357).

The fusion protein of the present disclosure can also be administeredwith agents that inhibit TGF-β signaling.

Additional agents that can be combined with a fusion protein describedherein include agents that enhance tumor antigen presentation, e.g.,dendritic cell vaccines, GM-CSF secreting cellular vaccines, CpGoligonucleotides, and imiquimod, or therapies that enhance theimmunogenicity of tumor cells (e.g., anthracyclines).

Another therapy that can be combined with the fusion protein describedherein is a therapy that inhibits a metabolic enzyme such as indoleaminedioxigenase (IDO), dioxigenase, arginase, or nitric oxide synthetase.

Another class of agents that can be used with the fusion proteindescribed herein includes agents that inhibit the formation ofadenosine, e.g., CD73 inhibitors, or inhibit the adenosine A2A receptor.

Other therapies that can be combined with the fusion protein describedherein for treating a disease or disorder, e.g., cancer, includetherapies that reverse/prevent T cell anergy or exhaustion and therapiesthat trigger an innate immune activation and/or inflammation at a tumorsite.

Other therapies that can be combined with the fusion protein describedherein for treating a disease or disorder, e.g., cancer, includetherapies that block IL-8, e.g., with HuMax-IL8.

A fusion protein described herein can be combined with more than oneimmuno-oncology agent, and can be, e.g., combined with a combinatorialapproach that targets multiple elements of the immune pathway, such asone or more of the following: a therapy that enhances tumor antigenpresentation (e.g., dendritic cell vaccine, GM-CSF secreting cellularvaccines, CpG oligonucleotides, imiquimod); a therapy that inhibitsnegative immune regulation e.g., by inhibiting CTLA-4 and/orPD1/PD-L1/PD-L2 pathway and/or depleting or blocking Tregs or otherimmune suppressing cells; a therapy that stimulates positive immuneregulation, e.g., with agonists that stimulate the CD-137, OX-40, and/orCD40 or GITR pathway and/or stimulate T cell effector function; atherapy that increases systemically the frequency of anti-tumor T cells;a therapy that depletes or inhibits Tregs, such as Tregs in the tumor,e.g., using an antagonist of CD25 (e.g., daclizumab) or by ex vivoanti-CD25 bead depletion; a therapy that impacts the function ofsuppressor myeloid cells in the tumor; a therapy that enhancesimmunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell orNK cell transfer including genetically modified cells, e.g., cellsmodified by chimeric antigen receptors (CAR-T therapy); a therapy thatinhibits a metabolic enzyme such as indoleamine dioxigenase (IDO),dioxigenase, arginase, or nitric oxide synthetase; a therapy thatreverses/prevents T cell anergy or exhaustion; a therapy that triggersan innate immune activation and/or inflammation at a tumor site;administration of immune stimulatory cytokines; or blocking of immunorepressive cytokines.

Fusion proteins described herein can be used together with one or moreof agonistic agents that ligate positive costimulatory receptors,blocking agents that attenuate signaling through inhibitory receptors,antagonists, and one or more agents that increase systemically thefrequency of anti-tumor T cells, agents that overcome distinct immunesuppressive pathways within the tumor microenvironment (e.g., blockinhibitory receptor engagement (e.g., PD-L1/PD-1 interactions), depleteor inhibit Tregs (e.g., using an anti-CD25 monoclonal antibody (e.g.,daclizumab) or by ex vivo anti-CD25 bead depletion), inhibit metabolicenzymes such as IDO, or reverse/prevent T cell anergy or exhaustion) andagents that trigger innate immune activation and/or inflammation attumor sites.

In some embodiments, the fusion protein of the present disclosure isadministered to a subject together with a BRAF inhibitor if the subjectis BRAF V600 mutation positive.

For example, the fusion protein of the present disclosure andcombination therapies described herein can be used in combination (e.g.,simultaneously or separately) with an additional treatment, such asirradiation and/or chemotherapy, e.g., using camptothecin (CPT-11),5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel,gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol),doxorubicin, or camptothecin+apo21/TRAIL (a 6× combo)), one or moreproteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2inhibitors (e.g., BH3I-2′ (bcl-xl inhibitor), indoleamine dioxygenase-1inhibitor (e.g., INCB24360, indoximod, NLG-919, or F001287), AT-101(R-(−)-gossypol derivative), ABT-263 (small molecule), GX-15-070(obatoclax), or MCL-1 (myeloid leukemia cell differentiation protein-1)antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g.,smac7, smac4, small molecule smac mimetic, synthetic smac peptides (seeFulda et al., Nat Med 2002; 8:808-15), ISIS23722 (LY2181308), orAEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20antibodies (e.g., rituximab), angiogenesis inhibitors (e.g.,bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g.,Avastin), synthetic triterpenoids (see Hyer et al, Cancer Research 2005;65:4799-808), c-FLIP (cellular FLICE-inhibitory protein) modulators(e.g., natural and synthetic ligands of PPARy (peroxisomeproliferator-activated receptor γ), 5809354 or 5569100), kinaseinhibitors (e.g., Sorafenib), Trastuzumab, Cetuximab, Temsirolimus, mTORinhibitors such as rapamycin and temsirolimus, Bortezomib, JAK2inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, Lenalildomide, GSK3Pinhibitors, IAP inhibitors and/or genotoxic drugs.

The fusion protein of the present disclosure and combination therapiesdescribed herein can further be used in combination with one or moreanti-proliferative cytotoxic agents. Classes of compounds that can beused as anti-proliferative cytotoxic agents include, but are not limitedto, the following:

Alkylating agents (including, without limitation, nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes):Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN®) fosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists,pyrimidine analogs, purine analogs and adenosine deaminase inhibitors):Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for combining with the fusion proteinof the present disclosure, without limitation, taxanes, paclitaxel(paclitaxel is commercially available as TAXOL™), docetaxel,discodermolide (DDM), dictyostatin (DCT), Peloruside A, epothilones,epothilone A, epothilone B, epothilone C, epothilone D, epothilone E,epothilone F, furanoepothilone D, desoxyepothilone Bl,[17]-dehydrodesoxyepothilone B, [18]dehydrodesoxyepothilones B,C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A,trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D,16-desmethylepothilone B, epothilone BIO, discoderomolide, patupilone(EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (Discodermolide),TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), HalichondrinB, Eribulin mesylate (E-7389), Hemiasterlin (HTI-286), E-7974,Cyrptophycins, LY-355703, Maytansinoid immunoconjugates (DM-1), MKC-1,ABT-751, T1-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731,STA-5312, eleutherobin,17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,cyclostreptin, isolaulimalide, laulimalide,4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, andcryptothilone 1, in addition to other microtubuline stabilizing agentsknown in the art.

In cases where it is desirable to render aberrantly proliferative cellsquiescent in conjunction with or prior to treatment with the fusionprotein of the present disclosure described herein, hormones andsteroids (including synthetic analogs), such as 17a-Ethinylestradiol,Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone,Dromostanolone propionate, Testolactone, Megestrolacetate,Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolone,Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine,Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, ZOLADEX®,can also be administered to the patient. When employing the methods orcompositions described herein, other agents used in the modulation oftumor growth or metastasis in a clinical setting, such as antimimetics,can also be administered as desired.

In some embodiments, the combination of the fusion protein of thepresent disclosure and a second agent discussed herein can beadministered concurrently as a single composition in a pharmaceuticallyacceptable carrier, or concurrently as separate compositions with thefusion protein of the present disclosure and the second agent in apharmaceutically acceptable carrier. In some embodiments, thecombination of the fusion protein of the present disclosure and thesecond agent can be administered sequentially. The administration of thetwo agents can start at times that are, e.g., 30 minutes, 60 minutes, 90minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48hours, 3 days, 5 days, 7 days, or one or more weeks apart, oradministration of the second agent can start, e.g., 30 minutes, 60minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours,36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks afterthe first agent has been administered.

In some embodiments, an anti-neoplastic antibody that can be combinedwith a fusion protein of the present disclosure and/or a second agentincludes RITUXAN® (rituximab), HERCEPTIN® (trastuzumab), BEXXAR®(tositumomab), ZEVALIN® (ibritumomab), CAMPATH® (alemtuzumab),LYMPHOCIDE® (eprtuzumab), AVASTIN® (bevacizumab), and TARCEVA®(erlotinib), or any combination thereof. In some embodiments, the secondantibody useful for the combination therapy with a fusion protein of thepresent disclosure can be an antibody drug conjugate.

In some embodiments, a fusion protein of the present disclosure alone orin combination with another agent is used concurrently or sequentiallywith bone marrow transplantation to treat a variety of tumors ofhematopoietic origin.

Provided herein are methods for altering an adverse event associatedwith treatment of a hyperproliferative disease (e.g., cancer) with animmuno stimulatory agent, comprising administering a fusion protein ofthe present disclosure with or without a second agent, to a subject. Forexample, the methods described herein provide for a method of reducingthe incidence of immuno stimulatory therapeutic antibody-induced colitisor diarrhea by administering a non-absorbable steroid to the patient. Asused herein, a “non-absorbable steroid” is a glucocorticoid thatexhibits extensive first pass metabolism such that, following metabolismin the liver, the bioavailability of the steroid is low, i.e., less thanabout 20%. In some embodiments described herein, the non-absorbablesteroid is budesonide. Budesonide is a locally-actingglucocorticosteroid, which is extensively metabolized, primarily by theliver, following oral administration. ENTOCORT EC® (Astra-Zeneca) is apH- and time-dependent oral formulation of budesonide developed tooptimize drug delivery to the ileum and throughout the colon. ENTOCORTEC® is approved in the U.S. for the treatment of mild to moderateCrohn's disease involving the ileum and/or ascending colon. In someembodiments, a fusion protein of the present disclosure in conjunctionwith a non-absorbable steroid can be further combined with a salicylate.Salicylates include 5-ASA agents such as, for example: sulfasalazine(AZULFIDINE®, Pharmacia & Up John); olsalazine (DJPENTUM®, Pharmacia &Up John); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.); andmesalamine (ASACOL®, Procter & Gamble Pharmaceuticals; PENTASA®, ShireUS; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay).

7.8 Kits

As used herein, a kit comprises an IL2/IL2Rα fusion protein for use inmodulating the immune response, as described elsewhere herein. The terms“kit” and “system,” as used herein are intended to refer to at least oneor more IL2/IL2Rα fusion protein which, in specific embodiments, are incombination with one or more other types of elements or components(e.g., other types of biochemical reagents, containers, packages, suchas packaging intended for commercial sale, instructions of use, and thelike).

In some embodiments, disclosed is a kit comprising (a) one or more of anIL2/IL2Rα fusion protein as described herein, a composition comprisingan IL2/IL2Rα fusion protein as described herein, a nucleic acid encodingfor an IL2/IL2Rα fusion protein as described herein, a vector, and/or ahost cell; and (b) and instructions for administering the fusion proteinto a subject in need thereof. In some embodiments, disclosed is a kitcomprising (a) an IL2/IL2Rα fusion protein as described herein and (b)and instructions for administering the fusion protein to a subject inneed thereof. In some embodiments, disclosed is a kit comprising (a) acomposition comprising an IL2/IL2Rα fusion protein as described hereinand (b) and instructions for administering the composition to a subjectin need thereof. In some embodiments, disclosed is a kit comprising (a)a nucleic acid encoding for an IL2/IL2Rα fusion protein as describedherein and (b) and instructions for administering the nucleic to asubject in need thereof. In some embodiments, disclosed is a kitcomprising (a) a vector as described herein and (b) and instructions foradministering the vector to a subject in need thereof. In someembodiments, disclosed is a kit comprising (a) a host cell as describedherein and (b) and instructions for administering the host cell to asubject in need thereof.

In a specific embodiment, provided herein is a pharmaceutical pack orkit comprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions described herein, such asone or more fusion proteins provided herein. In some embodiments, thekits contain a pharmaceutical composition described herein and anyprophylactic or therapeutic agent, such as those described herein. Incertain embodiments, the kits may contain a T cell mitogen, such as,e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA),or a TCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody. Optionally associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

Also provided herein are kits that can be used in the above methods. Inone embodiment, a kit comprises a fusion protein described herein,preferably a purified fusion protein, in one or more containers. In aspecific embodiment, kits described herein contain a substantiallyisolated fusion protein as a control. In another specific embodiment,the kits described herein further comprise a control antibody or fusionprotein which does not react with an IL2 and/or IL2-Rα antigen. Inanother specific embodiment, kits described herein contain one or moreelements for detecting the binding of the fusion protein to an IL2and/or IL2-Rα antigen (e.g., the fusion protein can be conjugated to adetectable substrate such as a fluorescent compound, an enzymaticsubstrate, a radioactive compound or a luminescent compound, or a secondantibody which recognizes the first antibody can be conjugated to adetectable substrate). In specific embodiments, a kit provided hereincan include a recombinantly produced or chemically synthesized fusionprotein. The antigen to a fusion protein disclosed herein as provided inthe kit can also be attached to a solid support. In a more specificembodiment, the detecting means of the above described kit includes asolid support to which an antigen of the fusion protein is attached.Such a kit can also include a non-attached reporter-labeled anti-humanantibody or anti-mouse/rat antibody. In this embodiment, binding of thefusion protein to an antigen can be detected by binding of the saidreporter-labeled antibody.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986)); Crooks, Antisense drug Technology:Principles, strategies and applications, 2^(nd) Ed. CRC Press (2007) andin Ausubel et al. (1989) Current Protocols in Molecular Biology (JohnWiley and Sons, Baltimore, Md.).

All of the references cited above, as well as all references citedherein and the amino acid or nucleotide sequences (e.g., GenBank numbersand/or Uniprot numbers), are incorporated herein by reference in theirentireties.

The following examples are offered by way of illustration and not by wayof limitation.

8. EXAMPLES Example 1. IL2-CD25 Fusion Protein Forms a Stable,Homogeneous Homo-Dimer

The size and oligomeric state of the fusion proteins were studied bysize-exclusion chromatography coupled to an in-line multi-angle lightscattering detector (SEC-MALS). Samples were prepared by injecting 30 μgof stock protein sample. Isocratic separations were performed on a GEHealthcare Superdex 200 Increase 10/300 GL column (10 mm×300 mm)connected to a Prominence Shimadzu UFLC system consisting of a degasser,isocratic pump, chilled sample holder with injector, UV/vis detector,and column oven in buffer containing 40 mM Tris, 200 mM NaCl (pH 7.5),with 0.02% Na azide added and 0.1 um filtered running at a flow rate of0.75 mL/min. Samples were injected onto the column using an Shimadzuautosampler, and data were obtained from three online detectorsconnected in series: a Shimadzu SPD-20 dual wavelength UV/visspectrophotometer set for collection at 280 nm, followed by a WyattTechnologies mini-Dawn TREOS three angle laser light scattering detectorand then a Wyatt Optilab T-rEX interferometric refractometer. Data werecollected and analyzed using Astra 6 (Wyatt) and LabSolutions Lite(Shimadzu) software.

The data in FIG. 1 show typical absolute mass versus elution time fromanalytical size exclusion chromatography. The sample analyzed in FIG. 1is for IL2-CD25(22-212) (SEQ ID NO: 16) fused to a His tag (GGHHHHHH(SEQ ID NO: 100), which has a theoretical molecular mass of 37,812(reduced) for the polypeptide chain. The data indicate thatIL2-CD25(22-212) forms a homogeneous species across the elution profilehaving a measured absolute mass of 93 kDa. The elution peak shows noevidence of monomeric species nor of oligomeric species of higher orderthan the main peak. The mass value of 93 kDa indicates the moleculeforms a homodimer and is also glycosylated (about 18% mass from known N-and O-linked glycosylation sites on IL2 and CD25).

Example 2: IL2-CD25 Fusion Proteins Bind Equivalently to sCD25 andsIL2Rβ/IL2Rγ Heteroreceptors with Attenuated Observed Affinity

Surface plasmon resonance (SPR) studies were performed on a Biacore T100and/or T200 instrument (GE Healthcare) at 25° C. The binding of thefusion protein analytes were tested in phosphate buffered saline (PBS-T)(pH 7.1) on surfaces consisting of a low density (˜300RU) ofbiot-hCD25-BioP-TVMV-His which had been His cleaved (hCD25) and capturedon a streptavidin, SA sensor chip or hCD122(27-241)-hFc-D/hCD132(23-263)-hFc-K heterodimeric Fc fusion (hIL2-Rb/g) that had beencaptured via Protein A immobilized CM5 sensorchip surface using standardethyl(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (NHS)chemistry, with ethanolamine blocking. The protein analytes wereinjected in a titration series and regeneration back to baseline wasperformed by 2×8s injections of low pH buffer. The data were analyzedusing the Biacore T-200 Evaluation software.

FIG. 2 shows prototypical binding of the IL2-CD25 fusion protein (SEQ IDNO: 16) to human sCD25. The observed apparent equilibrium dissociationconstant of 4.2 micromolar is approximately 150-fold weaker than theaffinity of isolated IL2 to sCD25 as measured by surface plasmonresonance (Liparoto, S. F., Myszka, D. G., Wu, Z., Goldstein, B., Laue,T. M., and Ciardelli, T. L. (2002) Biochemistry 41, 2543-51).

TABLE 9 Pharmacokinetics of binding of the IL2-CD25 fusion protein (SEQID NO: 16) to human sCD25. ka (1/Ms) kd (1/s) KD (M) 1.29E+4 0.0544.2E−6

Table 10 below shows prototypical binding observed equilibrium K_(D)values for short and long versions of IL2-CD25 to human sCD25 andsIL2Rβ/IL2Rγ heterodimer. The observed apparent equilibrium dissociationconstants for the various IL2-CD25 fusion proteins are markedlyattenuated compared to IL2. Binding to sCD25 is approximately 100-foldweaker for the IL2-CD25 fusions compared to IL2. Likewise binding of theIL2-CD25 fusions is about 150-fold weaker to the beta-gamma heterodimerthan is IL2.

TABLE 10 Observed equilibrium K_(D) values for binding IL2 and IL2-CD25constructs to sCD25 or sIL2Rβ/IL2Rγ heterodimer as measured by surfaceplasmon resonance. K_(D) to K_(D) sIL2Rβ/ to IL2Rγ sCD25, hetero-Receptor μM dimer, μM IL2 0.025 0.0004 IL2-CD25(22-187) 1.6 0.054IL2-CD25(22-212) 2.3 0.065 IL2-CV-CD25(22-240) 2.1 0.063IL2-CD25(22-240) 2.4 0.044 Fc1.1-7linker-IL2-CD25(22-212) 5.6 N.D.Bivalent SEQ ID NO: 67 IL2-CD25(22-212)-Fc1.1 2.5 0.1 SEQ ID NO: 68IL2-T3A-CD25(22-187)-N89Q-T95A-T106A 1.9 N.D. (SEQ ID NO: 202)Fc-(IL2(V91K)CD25)2 bivalent 3.0 SEQ ID NO: 203HSA-(G4S)3-IL2-CD25(22-187) 4.0 0.03 SEQ ID NO: 19 IL2(C145A)-CD25(22-212)-GG-HSA 2.1 0.03 SEQ ID NO: 20 HumanFc1.1(f)-AZ1-IL2(C145S)-CD25(22-187)-G 3.0 0.15 SEQ ID NO: 26IL2(C145S)-CD25(22-187)-HuFc1.1(f)-AZ1 2.0 0.15 SEQ ID NO: 27IL2(V111K)-CD25 1.9 SEQ ID NO: 69 IL2(D40T)-CD25 2.8 SEQ ID NO: 70

Example 3: Truncated Versions of IL2-CD25 have Improved StabilityAgainst Aggregation: Accelerated Stability Testing

The stability of the fusion proteins were tested by incubating at 40° C.in buffers of pH range 4-8. Fusion proteins were concentrated to about15 mg/ml and dialyzed at 4° C. into the following buffers: 1) 20 mMAcetate, 250 mM Sucrose, pH 4, 2) 20 mM Citrate, 250 mM Sucrose, pH 5,3) 20 mM Histidine, 250 mM Sucrose, pH 6, 4) 20 mM Phosphate, 250 mMSucrose, pH 7 and 5) 20 mM Tris, 250 mM Sucrose, pH 8.4 (roomtemperature). After recovery from dialysis, the concentration of thefusion proteins were normalized to 10 mg/ml by dilution with dialysisbuffer, and placed in an incubator monitored at 40° C. for four weeks,with aliquots removed just prior to incubation (t0), after one week (1w)and after four weeks (4w) of 40° C. incubation. Each time point wasanalyzed by Size Exclusion Chromatography on a Shodex KW403-4F column,connected to an Agilent 1260 HPLC system in buffer containing 100 mMSodium Phosphate, 150 mM Sodium Chloride, pH7.3 (0.2 μm filtered)running at a flow rate of 0.30 mL/min.

As seen in Tables 11 and 12, the truncated versions of IL2-CD25 displaymuch better high molecular weight and low molecular weight profilesafter accelerated stability studies than the longer versions. This isespecially true for pH values 4, 5, 6 and 7. Samples were held atdifferent pH conditions for four weeks and forty degrees Celsius. Theresults shown as percent of high molecular weight and low molecularweight fractions of each construct indicate greatly improved stabilityof the short constructs compared to the long ones. The results shown aspercent of main peak fractions of each construct indicate greatlyimproved stability of the short constructs compared to the long ones.

TABLE 11 Accelerated stability profiles of IL2-CD25 fusion proteins.IL2-CD25(22-187) IL2-CD25(22-212) IL2-C145V-CD25(22-240)IL2-CD25(22-240) HMW LMW HMW LMW HMW LMW HMW LMW pH (%) (%) (%) (%) (%)(%) (%) (%) 4 2.3 6.3 5.3 3.4 19.4 5.7 13.0 13.4 5 1.9 3.9 1.8 1.9 12.50.3 14.3 2.6 6 1.3 2.2 1.9 1.2 13.3 0.0 15.9 0.0 7 5.1 3.8 2.6 2.5 9.42.3 10.5 2.5 8 5.5 4.2 2.6 2.7 6.2 2.3 7.8 2.7The IL2-CD25 fusion proteins herein contain a His tag (GGHHHHHH, SEQ IDNO: 100) for purpose of easy purification.

TABLE 12 Accelerated stability profiles of IL2-CD25 fusion proteins.IL2-CD25 IL2-CD25 IL2-C145V-CD25 IL2-CD25 pH (22-187) (22-212) (22-240)(22-240) 4 91.4 91.3 74.9 73.6 5 94.2 96.4 87.2 83.1 6 96.6 97.0 86.784.1 7 91.1 94.9 88.3 87.0 8 90.3 94.7 91.6 89.5

Example 4: Pharmacokinetic Studies in Non-Human Animals

Pharmacokinetic Studies

All animal protocols were approved by Central New Jersey InstitutionalAnimal Care and Use Committee and animals were housed according toguidelines. Female Balb/C mice weighing 19-20 grams were purchased fromCharles-River (Wilmington, Mass.). Non-fasted Balb/C mice wereadministered a single 0.5 mg/kg dose of the indicated molecule, eitherby intravenous (IV) route via tail-vein or subcutaneous (SC) route.IL2(21-153)-(G3S)3-CD25(22-240) used in pharmacokinetic studies has aHis6-tag. Blood was collected from tail vein at following time-pointsafter dose: 5 minutes (IV only), 1h, 7h, 24h, 48h, 72h, 96h and 168h.For monkey PK study, male cynomolgus monkeys were obtained fromBuckshire Corporation (Perkasie, Pa.). Monkeys (N=3, average body weight7.8 kg) were administered a single 0.075 mg/kg subcutaneous dose ofhIL2-CD25 (22-212). Serial blood samples were collected from a femoralartery from conscious and chaired monkeys at 5h, 24h, 48h, 72h, 96h,168h, 192h, and 240h following dosing. Blood samples were allowed tocoagulate and centrifuged at 4° C. (1500-2000×g) to obtain serum. Serumsamples were stored at −80° C. until further analysis.

Detection of Fusion Protein in Serum:

A ligand binding assay was used to detect levels of the fusion proteinin mouse or monkey serum samples. Briefly, 96-well Nunc MaxdiSorp flatbottom plates (Thermo Fisher Scientific, Denmark) were coated withcapture reagent (rat anti-human IL2 antibody, monoclonal clone:MQ1-17H12; Thermo Fisher Scientific, San Diego, Calif.), at 2 μg/mL inPBS overnight at 4° C. Plates were blocked with blocking buffer (5% BSAin PBS) and incubated at 25° C. for one hour. The standards, QCs andsamples were diluted 20-fold with assay buffer (PBS with 1% BSA and0.05% Tween 20) and added to the washed plates in duplicate, andincubated at 4° C. overnight. Washed plates were incubated withdetection reagent (human CD25 biotinylated antibody, R&D Systems,Minneapolis, Minn. at 250 ng/mL in assay buffer) and incubated for 2hours at 25° C. To the washed plates were added sequentially, at 25° C.,NeutrAvidin-HRP (Thermo Scientific, Rockford, Ill.) 100 ng/mL in assaybuffer for 45 minutes followed by chemiluminescent substrate mix (ThermoScientific, Rockford, Ill.) for one minute before reading in SpectraMaxplate reader at luminescence mode. Concentration of the analyte in serumsamples was calculated using the standard curve made by the Log-Loglinear curve fit algorithm of softmax analysis program (MolecularDevices).

TABLE 13 Pharmacokinetic parameters of indicated molecules in Balb/Cmice. AUC Bioavail- Dose CL Vss (0-last) Cmax Tmax T½ ability mg/kgRoute ml/h/kg ml/kg nM*h nM h h % IL2(21-153)- 0.5 IV 1.3  46.3 8046.3NA NA 19.4 NA (G3S)3-CD25(22- 240) 0.5 SC NA NA 3639.6 79   24 31 45%IL2(21-153)- 0.5 IV 4    94.2 3053.1 NA NA 19.4 NA (G3S)3-CD25(22- 212)0.5 SC NA NA 2033.1 54.6  7 23 67% IL2(21-153)- 0.5 IV 23.1  200.4 610.4NA NA 11.6 NA (G3S)3-CD25(22- 187) 0.5 SC NA NA 261.3 13.2  7 10.9 43%Fc1.1-GSGGSGG- 0.5 IV  1.96 167.6 1442 NA NA 66.6 NA IL2(21-153)-(G3S)3-CD25(22- 212) 0.5 SC NA NA 886.5  12.02 24 68.8 61% IL2(21-153)-0.5 IV 1.2 194.4 1771.3 NA NA 120 NA (G3S)3-CD25(22- 212)-Fc1.1 0.5 SCNA NA 1478.6 16.2 24 61.3 83% NA = not applicable, ND = not determined.

TABLE 14 Pharmacokinetic parameters of hIL2- CD25 (22-212) in cynomolgusmonkeys. AUC Dose (0-last) Cmax Tmax T_(1/2) mg/kg Route nM*h nM h dayIL2(21-153)- 0.075 SC 1200 7.6 24 2.1 (G3S)3-CD25(22- 212)

Example 5: Activity of IL2-CD25 Fusion Protein on Kit225 Cells

The biological activity of the modified fusion proteins was determinedby measuring the ability to stimulate IL2R endogenously expressed on theT cell line, Kit225 (an IL2 dependent human T cell line from a patientwith T cell chronic lymphocytic leukemia with OKT3+, -T4+, -T8−phenotype). Activity was measured by the use of a reporter systemsensitive to the signaling cascade of IL2R. Kit225 human T cells stablyintegrated with the firefly luciferase reporter gene under the controlof the IFNγ activation sequence (IRF1-GAS-Luc) were grown in mediumcontaining RPMI+Glutamax, 10% heat-inactivated FBS, 20 ng/mL recombinantIL2 (Invitrogen PHC0023), 1% Pen/Strep and 0.7 mg/mL Geneticin. A dayprior to the assay, reporter cells were washed and re-suspended in assaymedium (Phenol-red-free RPMI+L-glutamine, 10% heat-inactivated FBS, 1%Pen/Strep) to remove IL2 present in the growth medium, then incubatedovernight at 37° C. On the day of the assay, IL2 molecules were seriallydiluted in the assay buffer for a 3-fold, 11-point concentrationresponse curve, various concentrations of IL2 molecules were added to aCulturPlate-384 assay plate (Perkin Elmer cat. no. 6007688) followed bythe addition of 70,000 cells/well. Assay plates were incubated for 5hours in a 37° C. CO₂ incubator then equilibrated to room temperaturefor 20 minutes before adding ONE-GLO™ substrate (Promega E6120). Plateswere sealed and luminescent signals were measured on a Perkin ElmerEnvision. 11 points of IL2 dose response luminescent signals wereplotted using GraphPad Priam. The EC50 is defined as the concentrationof test IL2 fusion proteins corresponding to 50% activation derived fromthe 11-point fitted curve as determined using a four-parameter logisticregression model. The potency of the fusion proteins on IL2R signalingin primary cells was determined by stimulation of either humanperipheral blood mononuclear cells (PBMCs) or whole blood with fusionproteins. Tyrosine phosphorylation of pSTAT5 is an immediate consequenceof IL2R signaling and was detected in various cell populations in eitherwhole blood or PBMCs by flow cytometry. Blood samples or PBMCs weretreated with serial dilutions of hIL2-CD25 fusion proteins for 15 min at37° C. After incubation, cells were fixed and blood lysed with Lyse/Fixbuffer for 10 min (BD Phosflow). Cells were washed twice and thenpermeabilized in ice-cold methanol on ice for 30 min, further washedtwice. Samples were then treated with Human Fc Block (ebioscience),followed by staining with labeled antibodies for CD3, CD4, CD8, CD25,pSTAT5, Foxp3, and CD56. All samples were then analyzed by flowcytometry.

In in vitro cell based assays, the fusion protein exhibited potency ofIL2 receptor activation which was significantly right shifted (poorerpotency) relative to wt IL2. This is consistent with the reducedapparent affinity of the fusion protein in the non-covalent dimericform. Addition of an Fc tail to the fusion protein did produce a furthershift to poorer potency by 20-30 fold. Removal of the His-tag ortruncation of the C terminus had minimal effect on the potency of thefusion proteins. Similar relative potency results were obtained in wholeblood.

TABLE 15A Activity of IL2-CD25 fusion protein on Kit225 cells. FusionProtein EC₅₀ (ng/ml) IL2 WT 0.41 N = 9 hIL2-CD25 (22-240)-His 138 n = 2hIL2-C145V-CD25 (22-240)-His 166 n = 2 hIL2-CD25(22-212)-His 210 n = 2hIL2-CD25(22-212) 139 n = 12 hIL2-CD25(22-187)-His 145 n = 2hIL2-CD25(22-187) 49 n = 2 Fc1.1-7linker-IL2-CD25(22-212 3414 n = 2(bivalent) hIL2-CD25(22-212)-Fc1.1 (bivalent) 3413 n = 2

TABLE 15B Activity of IL2-CD25 fusion protein on Kit225 cells. Ymax (%)Potency % of wt IL2 Protein SEQ EC50 ng/ml STDEV n response wt-hIL2 0.420.23 9 N/A hIL2-CD25(22- 16 139 112 12 103%  212) HSA-hIL2- 19 972 856 4104%  CD25(22-187) hIL2-CD25(22- 20 205 78 4 95% 187)-HSA Fc-hIL2- 26327 90 3 91% CD25(22-187) hIL2-CD25 27 257 95 3 93% (22-187)-FcFc-(hIL2-CD25 67 737 264 4 97% (22-187))2 bivalent hIL2-CD25 202 39.43.1 3 85% (quad aglycosylated)

The selectivity of hIL2-CD25 proteins including hIL2-CD25 (22-212) (SEQID NO:16) signaling on Treg over other cell types can be measured bymonitored phosphorylation of STAT5 after 15 min in a mixed cellpopulation including in PBMCs or whole blood (FIGS. 9A and 9B,respectively). Freshly acquired blood samples were treated with serialdilutions of hIL2-CD25 (22-212) (SEQ ID NO:16) for 15 min at 37° C.After incubation, cells were processed (fixed and permeabilized),stained with labeled antibodies for CD3, CD4, CD8, CD25, pSTAT5, Foxp3,and CD56, and analyzed by flow cytometry. Treg are very sensitive toIL2R stimulation by hIL2-CD25 (22-212) (SEQ ID NO:16) with maximalefficacy of 90% of Treg responding to stimulation by upregulation ofpSTAT5 and with a potency of 7+/−11 ng/ml. In contrast to the robustactivation observed on Treg, CD4 non-Treg (foxp3−), CD8 and NK cellswere less effectively stimulated by hIL2-CD25 (22-212) (SEQ ID NO:16);below 50% of these cells upregulated pSTAT5 even at concentrations up to2700 ng/ml on average across all other cell types (Table 16). Overallthe selectivity of IL2R on Treg over other cell types is maintained orincreased for hIL2-CD25 (22-212) (SEQ ID NO: 16) relative to rhIL2. Fc-or HSA-fusion proteins with IL2-CD25 maintained high Treg selectivity inwhole blood albeit with lower potency (Table 16).

TABLE 16 Potency and efficacy of stimulation of various cell types inhuman whole blood. Data is the average for 2-10 donors on freshly drawnblood from normal donors. The maximal % pSTAT5 positive cells wasdetermines at concentrations ~200-400x greater than the Treg potency.CD4 foxp3- CD8 T NK Treg Non-Treg cells cells Max @ Max @ Max @ Max @highest highest highest highest % pSTAT5 EC50 conc. conc. conc. conc.positive cells ng/ml tested tested tested tested hIL2-CD25 7.2 90% 48%27% 22% (22-212) (±10.4, (+/−8, (+/−14, (+/−11, (+/−19, (SEQ 16) n = 10)n = 10) n = 10) n = 10) n = 8) @2.7 μg/ @2.7 μg/ @2.7 μg/ ml ml mlFc-(IL2- 88 95% 50% 33% 8% CD25)₂ (±74, (+/−2, (n = 3) (n = 3) (n = 2)(SEQ 67) n = 4) n = 4) @24 μg/ @24 μg/ @24 μg/ bivalent ml ml mlFc-(IL2- 25 94% 60% 36% 6% CD25) (±12, (+/−1, (n = 3) (n = 3) (n = 3)(SEQ 26) n = 3) n = 3) @8.1 μg/ @8.1 μg/ @8.1 μg/ monovalent ml ml mlHSA-(G4S)3- 83 95% 59% 38% 6% IL2-CD25-FT (±53, (+/−3, (n = 2) (n = 2)(n = 2) (SEQ 19) n = 3) n = 3) @24 μg/ @24 μg/ @24 μg/ ml ml ml IL2-CD25FT- 43 92% 60% 39% 10% GG-HSA (±35, (+/−5, (n = 2) (n = 2) (n = 2) (SEQ20) n = 2) n = 2) @8.1 μg/ @8.1 g/ @8.1 μg/ ml ml ml (IL2-CD25)- 11.494% 67% 49% 25% Fc (SEQ 27) (±1.6, (+/−5, n = 2 n = 2 n = 2 monovalent n= 2) n = 2) @8.1 μg/ @8.1 μg/ @8.1 μg/ ml ml ml

Example 6: In Vivo Activity of Increasing Leukocytes by TruncatedIL2-CD25 Fusion Proteins

The ability of fusion proteins to increase leukocytes in vivo was testedin mice with a humanized immune system. 16-20 weeks old femaleNSG-huCD34 engrafted mice (NOD.Cg-Prkdc^(scid) IL2rg^(tm1Wjl)/SzJ, whichare reconstituted with human CD34+ hematopoietic stem cells) werepurchased from Jackson Laboratory. Each humanized mouse was examined forthe presence of hCD45+ cells and murine CD45+ cells (mCD45+) inperipheral blood by flow cytometry 14 weeks post engraftment. Mice wereselected with at least 25% human CD45 engraftments. The engrafted humanhematopoietic stem cells have been tested to be free of HIV, HBV, HCVand LCMV (lymphocytic choriomeningitis virus). Animals were providedstandard rodent chow and purified water ad libitum and acclimated for aminimum of 1 week prior to use. All procedures using animals werereviewed and approved by the BMS Institutional Animal Care and UseCommittee. Every third day, 16-20 weeks old NSG-huCD34 engrafted micewere dose subcutaneously either with PBS or with fusion proteinhuIL2-CD25(22-187), huIL2-CD25(22-212) and HuIL2-CD25(22-240) at 10μg/mouse in a volume of 200 μl via the sub-cutaneous while the animalswere under anesthesia. Total of three subcutaneous doses wereadministered on Day 0, 3 and 6. 24 hrs following the last dose, alltreated groups were anesthetized, spleen was harvested in HBSS for FACSanalysis of Treg, CD8 T, or NK cells. All three fusion proteins showedsimilar ability to increase spleen cell numbers.

Example 7: Stability of the (G3S)3 Peptide Linker inIL2(21-153)-(G3S)3-CD25(22-212) in Human or Mouse Serum

Liquid chromatography with tandem mass spectrometry (LC-MS/MS)-basedbioanalytical method was developed to support the evaluation ofstability of the (G3S)3 linker in in vitro serum stability studies andin vivo monkey PK study. Following capture ofIL2(21-153)-(G3S)3-CD25(22-212) (i.e., SEQ ID NO:16) using rat anti-IL-2antibody, the peak area ratio of two signature peptides, DLISNINVIVLELK(from the IL-2 domain of IL2(21-153)-(G3S)3-CD25(22-212)) andEPPPWENEATER (from the CD25 domain of IL2(21-153)-(G3S)3-CD25(22-212)),was used to evaluate the linker stability between IL-2 and CD25.Linker-cleavage and resulting circulating cleaved product in serum wouldresult in a systematic increase in the IL2/CD25 area ratio >1.

To test the linker-cleavage liability of IL2(21-153)-(G3S)3-CD25(22-212)in vitro, 0.5 μM of IL2(21-153)-(G3S)3-CD25(22-212) was incubated inhuman serum (Bioreclamation Cat# HMSRM-M; Lot# BRH1332647) or mouseserum (Bioreclamation Cat# MSESRM-BALB-M; Lot# MSE264349) in a totalvolume of 1.5 ml for up to 72 hours at 37° C. Samples (200 μL serum)were collected at 0, 4, 24, 48, and 72 hours and stored at −80° C. untilanalysis of peak area ratio by LC-MS/MS. To identify presence ofcirculating cleavage product due to linker-cleavage in vivo after asingle dose of IL2(21-153)-(G3S)3-CD25(22-212) in monkey, serum samplescollected at 5, 24, 48, 72, 96, and 168 hours after dose were analyzedfor peak area ratio by LC-MS/MS.

As shown in FIG. 10, the peak area ratio of signature peptides in IL2and CD25 domains of IL2(21-153)-(G3S)3-CD25(22-212) remained close tounity over 72 hours of in vitro incubation in human or mouse serum at37° C., therefore indicating negligible linker-cleavage inIL2(21-153)-(G3S)3-CD25(22-212) both in human serum and in mouse serum.

In addition, as shown in FIG. 11, in serum samples collected from themonkey after a single subcutaneous dose of 0.075 mg/kgIL2(21-153)-(G3S)3-CD25(22-212), ratio of IL2 and CD25 surrogatepeptides after immuno-capture using anti-IL2, remained close to unity.The data suggest absence of linker-cleavage in serum of monkeys dosedwith IL2(21-153)-(G3S)3-CD25(22-212) (i.e., SEQ ID NO:16).

What is claimed is:
 1. A fusion protein comprising: (a) a firstpolypeptide comprising an Interleukin-2 (IL2) polypeptide; and (b) asecond polypeptide comprising an extracellular domain of anInterleukin-2 Receptor alpha (IL2Rα) polypeptide; wherein (i) theextracellular domain of the IL2Rα polypeptide has at least one fewerglycosylation compared to the extracellular domain of native IL2Rα (SEQID NO:7); and/or (ii) the IL2 polypeptide has at least one fewerglycosylation compared to native IL2 (SEQ ID NO:2); and wherein thefusion protein has IL2 activity.
 2. The fusion protein of claim 1,wherein the extracellular domain of the IL2Rα polypeptide has at leastone fewer glycosylation, at least two fewer glycosylations, at leastthree fewer glycosylations, at least four fewer glycosylations, at leastfive fewer glycosylations, at least six fewer glycosylations, at leastseven fewer glycosylations, at least eight fewer glycosylations, or atleast nine fewer glycosylations compared to the extracellular domain ofnative IL2Rα (SEQ ID NO:7).
 3. The fusion protein of claim 1 or 2,wherein the IL2 polypeptide has at least one fewer glycosylationcompared to native IL2 (SEQ ID NO:2).
 4. The fusion protein of any oneof claims 1 to 3, wherein the first polypeptide comprises an amino acidsequence at least about 60%, at least about 70%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99% or about100% identical to SEQ ID NO:2.
 5. The fusion protein of any one ofclaims 1 to 4, wherein the second polypeptide comprises an amino acidsequence at least about 60%, at least about 70%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, orabout 100% identical to SEQ ID NO:12.
 6. The fusion protein of any oneof claims 1 to 5, wherein the extracellular domain of the IL2Rαpolypeptide having at least one fewer glycosylation comprises a mutationthat removes a glycosylation.
 7. The fusion protein of claim 6, whereinthe mutation removes an O-glycosylation and/or an N-glycosylation. 8.The fusion protein of claim 7, wherein the mutation removes anO-glycosylation.
 9. The fusion protein of claim 7, wherein the mutationremoves an N-glycosylation.
 10. The fusion protein of any one of claims6 to 9, wherein the mutation is a deletion of amino acids 167 to 219,amino acids 168 to 219, amino acids 169 to 219, amino acids 170 to 219,amino acids 171 to 219, amino acids 172 to 219, amino acids 173 to 219,amino acids 174 to 219, amino acids 175 to 219, amino acids 176 to 219,amino acids 177 to 219, amino acids 178 to 219, amino acids 179 to 219,amino acids 180 to 219, amino acids 181 to 219, amino acids 182 to 219,amino acids 183 to 219, amino acids 184 to 219, amino acids 185 to 219,amino acids 186 to 219, amino acids 187 to 219, amino acids 188 to 219,amino acids 189 to 219, amino acids 190 to 219, amino acids 191 to 219,or amino acids 192 to 219, corresponding to SEQ ID NO:7.
 11. The fusionprotein of claim 10, wherein the second polypeptide is SEQ ID NO:11. 12.The fusion protein of claim 10, wherein the second polypeptide is SEQ IDNO:12.
 13. The fusion protein of any one of claims 6 to 9, wherein themutation is one or more substitutions of an amino acid that isglycosylated with an amino acid that is not glycosylated.
 14. The fusionprotein of any one of claims 6 to 9, wherein the mutation is one or moresubstitutions of an amino acid that allows glycosylation at a nearbyamino acid with an amino acid that does not allow glycosylation at thenearby amino acid.
 15. The fusion protein of claim 13, wherein the oneor more substitutions are at amino acid N49, amino acid N68, amino acidT74, amino acid T85, amino acid T197, amino acid T203, amino acid T208,and amino acid T216, or any combination thereof, wherein the amino acidlocations correspond to SEQ ID NO:7.
 16. The fusion protein of claim 15,wherein the one or more substitutions are from asparagine to an aminoacid selected from the group consisting of alanine, threonine, serine,arginine, aspartic acid, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline,tryptophan, tyrosine, and valine.
 17. The fusion protein of claim 15,wherein the one or more substitutions are from threonine to an aminoacid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine, and valine.
 18. The fusionprotein of any one of claims 15 to 17, wherein one of the substitutionsis amino acid N49.
 19. The fusion protein of claim 18, wherein N49 ismutated to an amino acid selected from the group consisting of alanine,threonine, serine, arginine, aspartic acid, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, tryptophan, tyrosine, and valine.
 20. The fusionprotein of any one of claims 15 to 19, wherein one the substitutions isamino acid N68.
 21. The fusion protein of claim 20, wherein N68 ismutated to an amino acid selected from the group consisting of alanine,threonine, serine, arginine, aspartic acid, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, tryptophan, tyrosine, and valine.
 22. The fusionprotein of any one of claims 15 to 21, wherein one of the substitutionsis amino acid T74.
 23. The fusion protein of claim 22, wherein T74 ismutated to an amino acid selected from the group consisting of alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, tryptophan, tyrosine, and valine. 24.The fusion protein of any one of claims 15 to 23, wherein one of thesubstitutions is amino acid T85.
 25. The fusion protein of claim 24,wherein T85 is mutated to an amino acid selected from the groupconsisting of alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, tryptophan,tyrosine, and valine.
 26. The fusion protein of any one of claims 15 to25, wherein one of the substitutions is amino acid T197.
 27. The fusionprotein of claim 26, wherein T197 is mutated to an amino acid selectedfrom the group consisting of alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,tryptophan, tyrosine, and valine.
 28. The fusion protein of any one ofclaims 15 to 27, wherein one of the substitutions is amino acid T203.29. The fusion protein of claim 28, wherein T203 is mutated to an aminoacid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine, and valine.
 30. The fusionprotein of any one of claims 15 to 29, wherein one of the substitutionsis amino acid T208.
 31. The fusion protein of claim 30, wherein T208 ismutated to an amino acid selected from the group consisting of alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, tryptophan, tyrosine, and valine. 32.The fusion protein of any one of claims 15 to 31, wherein one of thesubstitutions is amino acid T216.
 33. The fusion protein of claim 32,wherein T216 is mutated to an amino acid selected from the groupconsisting of alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, tryptophan,tyrosine, and valine.
 34. The fusion protein of claim 14, wherein theone or more substitutions are at amino acid S50, amino acid S51, aminoacid T69, amino acid T70, amino acid C192, or any combination thereof,wherein the amino acid locations correspond to SEQ ID NO:7.
 35. Thefusion protein of claim 34, wherein one of the substitutions is at aminoacid S50.
 36. The fusion protein of claim 35, wherein S50 is mutated toproline.
 37. The fusion protein of any one of claims 34 to 36, whereinone of the substitutions is amino acid S51.
 38. The fusion protein ofclaim 37, wherein S51 is mutated to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan,tyrosine, and valine.
 39. The fusion protein of any one of claims 34 to38, wherein one of the substitution is amino acid T69.
 40. The fusionprotein of claim 39, wherein T69 is mutated to proline.
 41. The fusionprotein of any one of claims 34 to 40, wherein one of the substitutionsis amino acid T70.
 42. The fusion protein of claim 41, wherein T70 ismutated to an amino acid selected from the group consisting of alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, threonine, tryptophan, tyrosine, and valine. 43.The fusion protein of any one of claims 34 to 42, wherein one of thesubstitutions is amino acid C192.
 44. The fusion protein of claim 43,wherein C192 is mutated to an amino acid selected from the groupconsisting of alanine, arginine, asparagine, aspartic acid, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine.
 45. The fusion protein of any one of claims 1 to44, wherein the IL2 polypeptide having at least one fewer glycosylationcomprises a mutation that removes a glycosylation.
 46. The fusionprotein of claim 45, wherein the mutation is one or more substitutionsof an amino acid that is glycosylated with an amino acid that is notglycosylated.
 47. The fusion protein of claim 45, wherein the mutationis one or more substitutions of an amino acid that allows glycosylationat a nearby amino acid with an amino acid that does not allowglycosylation at the nearby amino acid.
 48. The fusion protein of claim46 or 47, wherein the one or more substitutions are from an alanine toan amino acid selected from the group consisting of arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, and valine.
 49. Thefusion protein of claim 46 or 47, wherein the one or more substitutionsare from a threonine to an amino acid selected from the group consistingof alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, tryptophan, tyrosine, and valine.50. The fusion protein of claim 46 or 47, wherein the one or moresubstitutions are from a cysteine to an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.
 51. The fusion protein of claim 50,wherein the one or more substitutions are from a cysteine to a serine.52. The fusion protein of claim 50, wherein the one or moresubstitutions are from a cysteine to an alanine.
 53. The fusion proteinof claim 50, wherein the one or more substitutions are from a cysteineto a valine.
 54. The fusion protein of claim 46 or 47, wherein the oneor more substitutions are at amino acid T3 compared to corresponding toSEQ ID NO:2.
 55. The fusion protein of any one of claims 47 to 54,wherein one of the substitutions is at amino acid C125.
 56. The fusionprotein of claim 55, wherein the substitution at amino acid C125 isselected from the group consisting of C125S, C125A, and C125V.
 57. Thefusion protein of claim 45, wherein the mutation is a deletion.
 58. Thefusion protein of claim 57, wherein the deletion is at amino acid A1.59. The fusion protein of any one of claims 1 to 58, wherein the fusionprotein is deglycosylated enzymatically or chemically.
 60. The fusionprotein of claim 59, wherein the fusion protein is deglycosylated byalkali, hydrazinolysis, PNGase F, Endo H, O-glycosidase, or anycombination thereof.
 61. The fusion protein of any one of claims 1 to60, further comprising a linker fused in frame between the firstpolypeptide and the second polypeptide.
 62. The fusion protein of claim61, wherein the linker is a glycine/serine linker.
 63. The fusionprotein of claim 62, wherein the glycine/serine linker comprises anamino acid sequence of (GS)_(n), (GGS)_(n), (GGGS)_(n), (GGGGS)_(n), or(GGGGS)_(n), wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or10.
 64. The fusion protein of claim 62, wherein the glycine/serinelinker comprises the amino acid sequence of (GGGS)₃.
 65. The fusionprotein of any one of claims 1 to 64, wherein the fusion protein furthercomprises a heterologous moiety fused to the first polypeptide and/orthe second polypeptide.
 66. The fusion protein of claim 65, wherein theheterologous moiety is a half-life extending moiety.
 67. The fusionprotein of claim 65 or 66, wherein the heterologous moiety comprises anon-polypeptide moiety.
 68. The fusion protein of claim 65 or 66,wherein the heterologous moiety comprises a polypeptide.
 69. The fusionprotein of claim 65, wherein the heterologous moiety comprises albumin,an immunoglobulin constant region or a portion thereof, animmunoglobulin-binding polypeptide, an immunoglobulin G (IgG),albumin-binding polypeptide (ABP), a PASylation moiety, a HESylationmoiety, XTEN, a PEGylation moiety, an Fc region, and any combinationthereof.
 70. The fusion protein of any one of claims 1 to 69, whereinthe fusion protein is more stable than the polypeptide consisting of SEQID NO:2 or SEQ ID NO:13.
 71. The fusion protein of claim 70, wherein thefusion protein has one or more properties selected from the groupconsisting of (i) increased thermodynamic stability compared to areference protein; (ii) increased TM compared to a reference protein;(iii) increased resistant to degradation compared to a referenceprotein; (iv) increased resistance to modifications compared to areference protein; (v) increased stability in vivo compared to areference protein; and (vi) any combination thereof, wherein thereference protein comprises (i) a first polypeptide comprising anInterleukin-2 (IL2) polypeptide; and (b) a second polypeptide comprisingan extracellular domain of an Interleukin-2 Receptor alpha (IL2Rα)polypeptide; and has at least one more glycosylation compared to thefusion protein.
 72. The fusion protein of any one of claims 1 to 71,which is a monomer.
 73. The fusion protein of any one of claims 1 to 72,which is a dimer.
 74. The fusion protein of claim 73, wherein the dimercomprises two monomers, and the monomers are associated with each othervia covalent bonds.
 75. The fusion protein of claim 73, wherein thedimer comprises two monomers, and the monomers are associated vianon-covalent bonds.
 76. The fusion protein of any one of claims 1 to 75,which has one or more pharmacokinetic properties selected from the groupconsisting of an increased half-life, increased C_(max), increased AUC,increased C_(min), decreased clearance, improved bioavailability, andany combination thereof, compared to the pharmacokinetic property of thepolypeptide consisting of SEQ ID NO:2 or SEQ ID NO:13.
 77. The fusionprotein of claim 76, which has an extended half-life.
 78. The fusionprotein of claim 77, wherein the extended half-life is at least about1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4fold, at least about 5 fold, at least about 6 fold, at least about 7fold, at least about 8 fold, at least about 9 fold, at least about 10fold, at least about 11 fold, at least about 12 fold, at least about 13fold, at least about 14 fold, at least about 15 fold, at least about 16fold, at least about 17 fold, at least about 18 fold, at least about 19fold, at least about 20 fold, at least about 21 fold, or at least about22 fold compared to the half-life of a polypeptide consisting of SEQ IDNO:2 or SEQ ID NO:13.
 79. A composition comprising the fusion protein ofclaim 72 and the fusion protein of claim
 73. 80. A nucleic acid thatencodes the fusion protein of any one of claims 1 to
 78. 81. A vectorcomprising the nucleic acid of claim
 80. 82. A host cell comprising thenucleic acid of claim
 80. 83. The host cell of claim 82, which is aeukaryotic cell.
 84. The host cell of claim 82, wherein the host cell isselected from the group consisting of a mammalian cell, an insect cell,a yeast cell, a transgenic mammalian cell, and a plant cell.
 85. Thehost cell of claim 84, wherein the host cell is a mammalian cell. 86.The host cell of claim 82, which is a prokaryotic cell.
 87. The hostcell of claim 86, wherein the prokaryotic cell is a bacterial cell. 88.A pharmaceutical composition comprising (a) the fusion protein of anyone of claims 1 to 78, the composition of claim 79, the nucleic acid ofclaim 80, the vector of claim 81, or the host cell of any one of claims82 to 87; and (b) a pharmaceutically acceptable excipient.
 89. A kitcomprising the fusion protein of any one of claims 1 to 78, thecomposition of claim 79, the nucleic acid of claim 80, the vector ofclaim 81, or the host cell of any one of claims 82 to 87 andinstructions for administering the fusion protein to a subject in needthereof.
 90. A method of producing the fusion protein of any one ofclaims 1 to 78, comprising: culturing the host cell of any one of claims82 to 87 under suitable conditions and recovering the fusion protein.91. The method of claim 90, wherein the host cell is a eukaryotic cellor a prokaryotic cell.
 92. The method of claim 90, wherein the host cellis a mammalian cell, an insect cell, a fungal cell, a plant cell, atransgenic mammalian cell, or a bacterial cell.
 93. The method of claim90, wherein the host cell is selected from the group consisting of a CHOcell, a HEK 293 cell, a NS0 cell, a Per C6 cell, a BHK cell, and a COScell.
 94. The method of claim 93, wherein the host cell is a bacterialcell.
 95. The host cell of claim 87 or method of claim 94, wherein thebacterial cell is Escherichia coli.
 96. A method of treating a diseaseor disorder a subject in need thereof, comprising administering to thesubject an effective amount of the fusion protein of any one of claims 1to 78, the composition of claim 79, the nucleic acid of claim 80, thevector of claim 81, the host cell of claim 82, or the pharmaceuticalcomposition of claim
 88. 97. The method of claim 96, wherein the diseaseor disorder is cancer.
 98. The method of claim 97, wherein the cancer isa bladder cancer, breast cancer, uterine cancer, endometrial carcinoma,ovarian cancer, colorectal cancer, colon cancer, head and neck cancer,lung cancer, stomach cancer, germ cell cancer, bone cancer, squamouscell cancer, skin cancer, neoplasm of the central nervous system,lymphoma, leukemia, sarcoma, virus-related cancer, small-cell lungcancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin'sor non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma,cervical cancer, ovarian cancer, liver cancer, myeloma, salivary glandcarcinoma, kidney cancer, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, or head or neck cancer, and any combinations thereof,
 99. Themethod of claim 96, wherein the disease or disorder is an inflammatorydisease or an autoimmune disease.
 100. The method of claim 99, whereinthe inflammatory disease or an autoimmune disease is selected from thegroup consisting of type 1 diabetes, multiple sclerosis, rheumatoidarthritis, celiac disease, systemic lupus erythematosus, lupusnephritis, cutaneous lupus, juvenile idiopathic arthritis, Crohn'sdisease, ulcerative colitis or systemic sclerosis, graft versus hostdisease, psoriasis, alopecia areata, HCV-induced vasculitis, Sjogren'ssyndrome, Pemphigus, Ankylosing Spondylitis, Behcet's Disease, Wegener'sGranulomatosis, Takayasu's Disease, Autoimmune Hepatitis, SclerosingCholangitis, Gougerot-sjögren, and Macrophage Activation Syndrome. 101.The method of claim 96, wherein the disease or disorder is an infectiousdisease.
 102. The method of claim 101, wherein the infectious disease iscaused by a pathogenic virus.
 103. The method of claim 102, wherein thepathogenic virus is selected from the group consisting of humanimmunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C,herpes virus, adenovirus, influenza virus, flaviviruses, echovirus,rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus,mumps virus, rotavirus, measles virus, rubella virus, parvovirus,vaccinia virus, human T-lymphotropic (HTL) virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JohnCunningham (JC) virus and arboviral encephalitis virus.
 104. The methodof claim 101, wherein the infectious disease is caused by pathogenicbacteria.
 105. The method of claim 104, wherein the pathogenic bacteriais selected from the group consisting of Chlamydia, rickettsialbacteria, mycobacteria, staphylococci, streptococci, pneumonococci,meningococci and gonococci, Klebsiella, Proteus, Serratia, Pseudomonas,Legionella, Diphtheria, Salmonella, bacilli, cholera, tetanus, botulism,anthrax, plague, leptospirosis, and Lymes disease bacteria.
 106. Themethod of claim 101, wherein the infectious disease is caused bypathogenic fungi.
 107. The method of claim 106, wherein the pathogenicbacteria is selected from the group consisting of Candida (albicans,krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans,Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia,rhizopus), Sporothrix schenkii, Blastomyces dermatitidis,Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasmacapsulatum.
 108. The method of claim 101, wherein the infectious diseaseis caused by pathogenic parasite.
 109. The method of claim 108, whereinthe pathogenic parasite is selected from the group consisting ofEntamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoebasp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii,Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosomacruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylusbrasiliensis.
 110. The method of any one of claims 96 to 109, furthercomprising administering to the subject a second agent.
 111. The methodof claim 110, wherein the second agent is a PD-1 antagonist, a CTLA-4antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, aLAG3 antagonist, or any combination thereof.
 112. The method of claim111, wherein the second agent is an anti-PD-1 antibody, an anti-PD-L1antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-KIRantibody, an anti-GITR antibody, an anti-LAG3 antibody, or anycombination thereof.
 113. The method of any one of claims 110 to 112,wherein the second agent is a cytokine inhibitor.
 114. The method ofclaim 113, wherein the cytokine inhibitor targets one or more of IL-6,IL-10, TGF-β, VEGF, IFN-γ, or any combination thereof.
 115. The methodof any one of claims 96 to 114, wherein the fusion protein isadministered via a topical, epidermal mucosal, intranasal, oral,vaginal, rectal, sublingual, topical, intravenous, intraperitoneal,intramuscular, intraarterial, intrathecal, intralymphatic,intralesional, intracapsular, intraorbital, intracardiac, intradermal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural or intrasternal route.